Ortho-diphenol profile and antioxidant activity of Algerian black olive cultivars: Effect of dry salting process

cti ye vers Teje Olive (Az or t yph ult we rous-chelating power. Among the o-diphenols identified in the salted olives, hydroxytyrosol was the most Relizane which contains the highest levels of polyphenols and o-diphenols exhibits the best antioxidant activity. Our results suggest that in addition to the processing, the cultivar and the geographical origin widely onditio ean co y 201 prevention of many diseases (Boskou et al., 2006). Among olive polyphenols, ortho-diphenols such as hydroxytyrosol, oleuropein and verbascoside (Ryan & Robards, 1998) are recognized as the most important in relation to their antioxidant activity which can be related to hydrogen donation, i.e., their ability to improve radical stability by forming an intra-molecular hydrogen bond be- tween the hydrogen of their hydroxyl group and their phenoxyl the organoleptic ished product. Ol- thod react al and p characteristics (Bianchi, 2003). For this, it would be interes monitor the qualitative and quantitative evolution of phen the table olives processed according to traditional methods. quently, the purpose of this study is to investigate the total poly- phenols, o-diphenol composition changes after dry salting of six black olive cultivars and to determine their antioxidant capacity in order to evaluate the effect of such process on the o-diphenol profile and antioxidant activity of the finished product, because of the limited data on this kind of processing.⇑ Corresponding author. Tel./fax: +213 34 21 47 62. E-mail address: [email protected] (H. Louaileche). Food Chemistry 157 (2014) 504–510 Contents lists available at ScienceDirect he lse tary components such as polyphenols might contribute to their protective role (Rice-Evans, Miller, & Paganga, 1997). The con- sumption of table olives provides a large amount of natural antiox- idants which play a major role in the antioxidant activity and in the tions of the phenolic profile. This affects both properties and the antioxidant capacity of the fin ive samples subjected to the same processing me ently, depending on their varietal, chemic http://dx.doi.org/10.1016/j.foodchem.2014.02.075 0308-8146/� 2014 Elsevier Ltd. All rights reserved. differ- hysical ting to olics of Conse- production was estimated to 192,785 ton in 2011 (ITAFV, 2011). The olives grown in Algeria belong to a wide range of cultivars including Azeradj, Bouchouk, Aberkane and Atefah. Numerous epidemiological surveys have shown an inverse rela- tionship between the intake of fruits and the incidence of coronary heart disease and certain cancers. Many constituents of these die- Greek-style naturally black olives and Californian-style black olives (Garrido-Fernández, Fernández-Díez, & Adams, 1997). However, there are some traditional preparations that have not attracted much attention. One of these involves the use of dry salt to elimi- nate the natural bitterness of the fruits and to make them edible. The processing of the raw olives causes considerable modifica- Cultivar Ortho-diphenol composition Antioxidant activity HPLC 1. Introduction The olive tree (Olea europaea) is gions of the world where climatic c those prevailing in the Mediterran crop area was around 188,923 ha b would have a pronounced influence on both o-diphenol composition and antioxidant activity of olives. � 2014 Elsevier Ltd. All rights reserved. cultivated in many re- ns are as favourable as untries. Algeria’s olive 1. The total table olive radicals (Visioli & Galli, 1998). Several studies have demonstrated that phenolic content in table olives depends on the processing method (Ben Othman, Roblain, Chammen, Thonart, & Hamdi, 2009; Bianchi, 2003; Romero et al., 2004). Three types of table ol- ives are of a great importance in the international trade and are mainly produced on an industrial scale: Spanish-style green olives, Keywords: Dry salting abundant followed by verbascoside and caffeic acid. The comparative study showed that Sigoise from Ortho-diphenol profile and antioxidant a cultivars: Effect of dry salting process Ouahiba Soufi a, Concepción Romero b, Louaileche Ha a Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, Uni b Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Avenida Padre García a r t i c l e i n f o Article history: Received 17 December 2013 Received in revised form 13 February 2014 Accepted 17 February 2014 Available online 26 February 2014 a b s t r a c t Six Algerian olive cultivars salting were investigated f dry salting affects total pol tively, depending on the c 10–35% for the reducing po Food C journal homepage: www.e vity of Algerian black olive tte a,⇑ ité de Bejaia, 06000 Bejaia, Algeria ro 4, 41012 Sevilla, Spain eradj, Sigoise, Bouchouk, Abelout, Aberkane and Atefah) processed by dry he total polyphenols, ortho-diphenol profile and antioxidant activity. The enol and o-diphenol contents with a loss rate of 6–46% and 7–50%, respec- ivar. Consequently, a decrease in the antioxidant activity was observed, r, 29–58% for the DPPH radical scavenging activity and 10–48% for the fer- mistry vier .com/locate / foodchem 2. Materials and methods 2.1. Olive samples Six black olive cultivars (Azeradj, Sigoise, Bouchouk, Abelout, Aberkane and Atefah) harvested at the fully ripe stage were hand-picked from different parts of olive trees on December 2010. Sigoise samples were harvested from three locations (Mas- cara, Relizane and Oran) (Table 1). 2.2. Processing of olive samples The collected olives (at least 2 kg) were treated with alternating (IKA model A 11 B, Staufen, Germany) and stored at �18 �C until phenolic content was expressed as mg of gallic acid equivalents results were expressed as mg of caffeic acid equivalents/100 g of dry weight (Bendini et al., 2003). 2.4.3. HPLC analysis of ortho-diphenols The preparation of extracts was based on the methodology pro- posed by Sánchez, Romero, Ramírez, and Brenes (2013). Freeze dried olive pulps (1 g) were homogenized with 6 mL of dimethyl- sulfoxide (DMSO). After stirring for 2 min, the mixture was centri- fuged at 28,000�g for 6 min at 22 �C; the supernatant was collected and filtered through a 0.22 lm nylon filter. An aliquot of filtrate (250 lL) was homogenized with 250 lL of internal stan- dard (syringic acid 0.2 mM in DMSO) and 500 lL of DMSO. A vol- ume of this mixture (20 lL) was injected for HPLC analysis; a percentage was raised to 40% in 10 min, which was maintained for 5 min. Finally, the methanol concentration for the last three Total phenolic compounds1 2716.15 ± 11.63aB 1601.77 ± 05.50bA 1296.47 ± 03.27aB 2154.29 ± 69.91bB 1103.63 ± 0.74dA 1022.12 ± 22.34cB ifica O. Soufi et al. / Food Chemistry 157 (2014) 504–510 505 Cultivar Origin Code Fresh olives Abelout Béjaia BT 2281.68 ± 14.62cA Aberkane Béjaia BK 2314.39 ± 68.45cA Azeradj Béjaia AZ 3782.02 ± 128.35bA Bouchouk Béjaia B 1197.18 ± 66.24eA Sigoise Mascara S1 3726.69 ± 73.43bA Sigoise Relizane S2 4355.02 ± 191.72aA Sigoise Oran S3 3662.41 ± 58.17bA Atefah Béjaia T 1684.11 ± 84.70dA A and B: within a row (effect of processing), different letters indicate statistically sign (GAE)/100 g of dry weight. 2.4.2. Total ortho-diphenol content A mixture of 2 mL of the olive extract and 500 lL of a 5% solu- tion of sodium molybdate, was shaken vigorously. After incubation for 15 min, the absorbance was measured at 370 nm and the Table 1 Total phenolic compounds and o-diphenols contents of studied olives. 2.4. Analysis of phenolic compounds 2.4.1. Total phenolic compounds The amount of total phenolics in extracts was determined according to the method of Kahkönen et al. (1999). Aliquots (200 lL) of extract were mixed with 1.0 mL of Folin–Ciocalteu re- agent and 800 lL of sodium carbonate (7.5%). After incubation for 30 min, the absorbance was measured at 725 nm (Uvi- mini1240 spectrophotometer, Shimadzu, Suzhou, China). The total analysis. 2.3. Extract preparation Freeze dried table olive pulps (100 mg) were homogenized in 10 mL of 50% acetone. After stirring for 30 min, the mixture was centrifuged (nüve NF 200, Ankara, Turkey) at 2800�g for 20 min. The supernatant was collected and filtered, and the residue was re-extracted. The filtered extracts were combined and washed with hexane (5 � 10 mL), then kept in refrigerator until analysis (McDonald, Prenzler, Antolovich, & Robards, 2001). layers of dry salt (0.8 kg), into baskets, and kept at room tempera- ture for 30–50 days depending on the cultivar (Panagou, 2006). The salting caused dehydration and the olives appear shriveled. The ob- tained olive pulps were freeze-dried (Christ, Alpha 1-4 LDplus, Osterode am Harz, Germany), then ground in electric blender a–h: Within a column, different letters indicate statistically significant differences (p < 0. 1 Results in mg GAE/100 g dw are expressed as the average ± Standard deviation of th 2 Results in mg caffeic acid/100 g dw are expressed as the average ± Standard deviatio 1217.10 ± 74.58efB 1026.50 ± 0.07fA 511.27 ± 01.82hB nt differences (p < 0.05). steps was increased to 60, 70, and 100% in 5 min periods. Initial conditions were reached in 15 min. An injection volume of 20 lL, a flow rate of 1 mL/min, and a temperature of 35 �C were used. Chromatograms were recorded at 280 nm (Romero, Brenes, García, & Garrido, 2002). The concentration of each compound was calculated using a standard curve. Hydroxytyrosol, oleuropein and verbascoside were purchased from Extrasynthese SA (Lyon Nord, Genay, France). Hydroxytyrosol-1-glucoside and caffeoyl ester were quantified using the response factors of hydroxytyrosol and caffeic acid, respectively. 2.5. Antioxidant activity 2.5.1. Reducing power The reducing power was estimated using the procedure de- scribed by Gülçin, Oktay, Küfreviog˘lu, and Aslan (2002). A volume of olive extract (250 lL) was mixed with 250 lL of phosphate buf- fer (0.2 M, pH 6.6) and 250 lL of potassium ferricyanide (1%). The mixture was incubated at 50 �C for 20 min. Aliquot (250 lL) of tri- chloroacetic acid (10%) and 200 lL of ferric chloride (0.1%) were added to the mixture. The absorbance was measured at 700 nm Total o-diphenols2 Salted olives Fresh olives Salted olives 1283.28 ± 53.02eB 1008.06 ± 09.49gA 625.84 ± 28.13fB 1548.48 ± 0.86dB 1043.26 ± 03.02eA 865.65 ± 19.80eB 2219.05 ± 102.32bB 2115.39 ± 11.62aA 1070.64 ± 07.72bB 1028.82 ± 59.04fA 845.67 ± 0.90hA 585.57 ± 03.59gB 2007.26 ± 91.21cB 1402.77 ± 1.31cA 899.99 ± 20.83dB flow rate of 1 mL/min and a temperature of 35 �C were used. The HPLC system consisted of a Waters 717 plus autosampler, a Waters 600 E pump, a Waters column heater module, and a Waters 996 photodiode array detector operated with Empower software (Waters Inc). A 25 cm � 4.6 mm i.d., 5 lm, Spherisorb ODS-2 (Waters Inc) column was used. The separation was achieved by gradient elution using an initial composition of 90% water (pH 2.5 adjusted with 0.15% phosphoric acid) and 10% methanol. The concentration of the latter solvent was increased to 30% in 10 min and maintained for 20 min. Subsequently, the methanol 05) between cultivars. ree replicates. n of three replicates. 5.5 Fr. Analysis of variance (ANOVA) was performed to estimate mist the statistically significant differences between olive samples for each parameter. P values 280 uk 0.27 ± 1.5 ± 0.0 .06ab .10d .03d .22c ree gluc mist which derived from hydrolysis of oleuropein. Furthermore, a quan- tity of verbascoside is hydrolyzed into caffeic acid and hydroxylty- rosol (Garrido-Fernández et al., 1997). However, we observed a decrease in hydroxytyrosol content for some cultivars (BT, B and S1) which might be explained by the oxidation of this compound during the dry salting. In the salted olives, the o-diphenol contents obtained by HPLC method were lower than those determined by spectrophotometric method (Tables 1 and 2). This can be attributed to the limited sen- sibility of spectrophotometric method due to the presence of inter- fering compounds such as proteins and sugars. This research finding is similar to those of Benmeddour, Mehinagic, Le Meurlay, and Louaileche (2013) and McDonald et al. (2001) who indicated that the level of total phenols determined by spectrophotometric method is not an absolute measurement of the amounts of pheno- lic materials but depends on their chemical reducing capacity rel- ative to an equivalent reducing capacity of used standard. This study demonstrated that the dry salting method affects the o-diphenol profile of olives which varied depending on the culti- vars. These variations might be related to the dry salting duration which depends on the cultivar. On the other hand, these com- pounds can be also oxidized when the period of the salting is pro- longed. In addition to the processing, other factors such as the geographical origin would have a pronounced influence on the to- Table 2 Individual ortho-diphenols1of fresh and salted black olives evaluated by HPLC-DAD at Abelout Aberkane Azeradj Boucho Fresh olives Hyd-1-Glu 12.85 ± 0.02d n.d. 69.63 ± 1.79b n.d. Hydroxytyrosol 27.48 ± 0.38a n.d. n.d. 15.39 ± Verbascoside 13.71 ± 0.18c 52.93 ± 0.38b 209.25 ± 1.74a 155.68 Oleuropein 395.81 ± 2.10de 505.27 ± 4.18d 1882.38 ± 31.76a 667.30 Caffeoyl ester 0.82 ± 0.02ef 1.95 ± 0.03cd n.d. 3.02 ± 0 Caffeic acid n.d. n.d. n.d. n.d. Salted olives Hyd-1-Glu n.d. n.d. n.d. n.d. Hydroxytyrosol 16.06 ± 0.69cd 15.58 ± 0.42cd 30.75 ± 0.88b 5.97 ± 0 Verbascoside n.d. 5.76 ± 0.06c 11.12 ± 0.27b 1.60 ± 0 Oleuropein n.d. n.d. n.d. n.d. Caffeoyl ester n.d. 0.49 ± 0.00b n.d. n.d. Caffeic acid n.d. 2.14 ± 0.04b 2.27 ± 0.19b 0.95 ± 0 1 Results are expressed in mg/100 g of dried weight ± standard deviation of th differences (ANOVA test, p < 0.05) between cultivars; Hyd-1-Glu (hydroxytyrosol-1- O. Soufi et al. / Food Che tal and individual o-diphenols since ‘‘S1’’, S2’’ and ‘‘S3’’ fresh olives had different o-diphenol profiles. 3.3. Antioxidant activity Several methods have been developed to measure the efficiency of dietary antioxidants. These methods focus on different mecha- nisms of the antioxidant defence system. In the present work, reducing power, antiradical activity and ferrous chelating activity were performed. 3.3.1. Reducing power An electron-donating reducing agent contributes to antioxidant activity by its capacity to donate an electron to free radicals, which results in neutralization of the radical. The dry salting had a signif- icant effect on reducing power of the studied olives. Among the fresh olives, Sigoise from Relizane exhibited the highest reducing power (5324 mg AAE/100 g). Along with the salted olives, Sigoise from Relizane had also the strongest reducing power (3914 mg AAE/100 g) followed by Sigoise from Oran (3426 mg AAE/100 g), then Azeradj (3093 mg AAE/100 g). The lowest reducing power was recorded for Bouchouk cultivar (1318 mg/100 g) (Fig. 1). The differences noted between the studied cultivars might be related to the phenolic content because of the role of such compounds in reducing capacity. The results showed that the decrease of the reducing power after processing varied from 10% (Azeradj) to 35% (Sigoise from Mascara). 3.3.2. DPPH free radical-scavenging activity The radical scavenging activity (RSA) assay constitutes cur- rently a method used to provide basic information on the antirad- ical activity of extracts. Among the fresh olives, Sigoise from Relizane exhibited the best antiradical activity (14,807 mg AAE/ 100 g) unlike Bouchouk cultivar (5448 mg AAE/100 g). After salt- ing, the strongest antiradical activity was also recorded for Sigoise from Relizane (8922 mg AAE/100 g), while the lowest one was ob- tained for Abelout cultivar (3334 mg AAE/100 g) (Fig. 2). A signifi- cant antiradical activity loss occurred after salting with variable values depending on the cultivars. The decrease rate ranged from 29% (B, S1 and S3) to 58% (BT). The scavenging effects of salted olive cultivars against the DPPH radical decreased in the following order: S2 > AZ = S3 = S1 > T > BKP BP BT (Fig. 2). This order is relatively similar to that ob- served in total phenolic and o-diphenol contents. These results could be explained by the amount and the type of phenolics, par- ticularly o-diphenols because of the importance of such com- nm. Sigoise (Mascara) Sigoise (Relizane) Sigoise (Oran) Atefah 164.98 ± 2.41a 45.12 ± 0.99cd n.d. 37.89 ± 0.28cd b 29.11 ± 0.37a 24.14 ± 0.17a n.d. n.d. 7c 201.15 ± 2.18a 74.89 ± 0.27b 18.00 ± 0.04c 8.97 ± 0.15c 2cd 999.24 ± 7.93b 320.65 ± 0.37e 264.04 ± 2.12e 902.78 ± 12.54bc 3.69 ± 0.09a 1.80 ± 0.05cd 1.43 ± 0.03de 2.52 ± 0.06bc n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 26.95 ± 0.01bc 95.43 ± 0.46a 24.25 ± 0.49bc n.d. 2.46 ± 0.01cd 18.55 ± 0.35a 4.34 ± 0.11cd n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.83 ± 0.00a 1.02 ± 0.04c 3.75 ± 0.27a n.d. n.d. replications; for each row, different letters (a–f) indicate statistically significant oside); n.d.: not detected. ry 157 (2014) 504–510 507 pounds in the antiradical activity of olive extracts. The individual o-diphenols have three hydroxyl groups bonded to the aromatic ring in an ortho position in relation to each other; this model of substitution seems to be the most important factor associated with a strong antiradical activity of olive extracts (Sroka & Cisowski, 2003; Visioli & Galli, 1998). This observation is in agreement with the results obtained for Sigoise from Relizane which presents the highest total and individual o-diphenol contents. 3.3.3. Ferrous chelating activity Ferrozine can quantitatively form complex with Fe2+. However, in the presence of chelating agents, the complex formation is dis- rupted. The fresh olives exhibited a considerable ferrous chelating activity which ranged from 25% (Atefah) to 51% (Sigoise from Reliz- ane). After salting, the activity decreased and the Sigoise from Relizane remained the strongest (41%), while the lowest activity was recorded for Bouchouk cultivar (11%) (Fig. 3). Ferrous-chelating activity of salted olive cultivars decreased in the following order: S2 (42%) > BK (35%) > BT = S3 (28%) > AZ (24%) > S1 (22%) > T (13%) > B (11%). This order differs from that of reducing power and antiradical activity. This can be explained mist A A A B B c 3000 4000 5000 6000 ow er (m g A A E /1 00 g) 508 O. Soufi et al. / Food Che by the mechanism involved in the ferrous chelating activity. The differences in the ferrous chelating activity noted between the investigated samples could be due to the structural properties of the antioxidant compounds present in olive extracts. Sigoise from Relizane (salted olives) which contains the highest levels of phen- olics exhibits the strongest ferrous chelating activity; this might be explained by the structure of phenolic compounds present in this cultivar, since the metal chelating ability depends on the number and position of the hydroxyl groups of phenolics (Wang et al., 2008). The present study demonstrates that the salting has a pro- nounced influence on the ferrous chelating activity; the decrease rate ranged from 10% (BK and BT) to 48% (B, S1 and T). A B g f B h 0 1000 2000 R ed uc in g p Cultiv Fig. 1. Reducing power of black olives cultivars (mg ascorbic acid equivalent/100 g dw). ( (p < 0.05). (a–h) Different letters indicate statistically significant differences (p < 0.05) be A A A A B e B d B b B d 0 2000 4000 6000 8000 10000 12000 14000 16000 A nt ir ad ic al a ct iv ity (m gA A E/ 10 0g ) Culti Fig. 2. Antiradical activity of black olive cultivars (mg ascorbic acid equivalent/100 g differences (p < 0.05). (a–e) Different letters indicate statistically significant differences A A A A B d B a B b B e Fresh Salted ry 157 (2014) 504–510 3.3.4. Relationship phenolic contents–antioxidant Activity The results indicated that all salted olive cultivars showed a lin- ear relationship with a positive correlation coefficient (p < 0.05) between total phenolics and antioxidant activity (Table 3) which was given by the reducing power (R = 0.94) and antiradical activity (R = 0.91). Statistically significant relationships were also observed be- tween total o-diphenol content and antioxidant activity which was given by the reducing power (R = 0.89) and the antiradical activity (R = 0.83). This indicates that overall, phenolic compounds and particularly o-diphenols play a major role in the antioxidant activity of olive extracts. On the contrary, the olive extracts exhib- ar A and B) Effect of salting: different letters indicate statistically significant differences tween cultivars. A A A A e B b B a B b B c var Fresh Salted dw). (A and B) Effect of salting: different letters indicate statistically significant (p < 0.05) between cultivars. B g mist A A A A B c B b B d 0 10 20 30 40 50 60 Fe +² C he la tin g ac tiv ity (% ) O. Soufi et al. / Food Che ited relatively moderate correlations between ferrous chelating activity with phenolic (R = 0.64) and o-diphenol contents (R = 0.74). This signifies that chelating activity could be partially related to the phenolic content, and that the antioxidant composi- tion has a major role in this activity. The results obtained for the antioxidant activity assessed by three assays showed that among the table olives studied, Sigoise from Relizane exhibits the best in vitro antioxidant activity and could be used as a source of natural antioxidants. 4. Conclusion This investigation has allowed the determination of black olive o-diphenol profile. The results of the present work denote that Algerian black olive cultivars might constitute a source of healthy compounds, especially o-diphenols which contribute to the antiox- idant capacity. The content of analyzed compounds depends on the cultivar and the geographical origin. The traditional method using Cu Fig. 3. Ferrous-chelating activity of black olive cultivars. (A and B) Effect of salting: dif letters indicate statistically significant differences (p < 0.05) between cultivars. Table 3 Correlation coefficients for the relationship between the antioxidant compounds and the antioxidant potential as measured by reducing power, DPPH and ferrous chelating activity. Correlation between Correlation coefficient (R)* Equation Total phenolic-reducing power 0.94 y = 1.45x � 40.95 Total phenolic-DPPH 0.91 y = 3.09x + 302.61 Total phenolic-ferrous chelating 0.64 y = 0.01x + 05.43 O-diphenols-reducing power 0.89 y = 2.99x � 53.88 O-diphenols-DPPH 0.83 y = 5.97x + 675.3 O-diphenols-ferrous chelating 0.74 y = 0.03x + 1.69 Verbascoside-reducing power 0.71 y = 80.83x + 2283 Verbascoside-DPPH 0.68 y = 175.0x + 5234 Verbascoside-ferrous chelating 0.70 y = 1.10x + 19.71 Hydroxytyrosol-reducing power 0.77 y = 21.51x + 2030 Hydroxytyrosol-DPPH 0.74 y = 49.26x + 4529 Hydroxytyrosol-ferrous chelating 0.69 y = 0.220x + 21.17 * Significant at P 6 0.05. dry salt affects significantly the total phenolic compounds, o-diphenol composition and the antioxidant activity of black ol- ives. However, it appears that despite of the phenolic and o-diphe- nol loss noted after dry salting, the antioxidant activity of salted olives remained considerable for some cultivars. Also, the table ol- ives obtained in a traditional way which does not required any alkaline reagent might have good organoleptic characteristics. The antioxidant activity of table olive extracts is related to the total phenolics; consequently, phenolic groups other than o-diphe- nols such as flavonoids can be involved in this activity. For this, it will be necessary to complete this study by identifying and quan- tifying the flavonoids of the investigated cultivars. ltivar ferent letters indicate statistically significant differences (p < 0.05). (a–g) Different A A A AB e B a B c B f Fresh Salted ry 157 (2014) 504–510 509 Acknowledgements Thanks are due to Ifri-Olive and Mr Ait Keddache S. and all per- sons who have provided the olive samples of this study and the Algerian Ministry of Higher Education and scientific Research who funded this work. 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Soufi et al. / Food Chemistry 157 (2014) 504–510 Ortho-diphenol profile and antioxidant activity of Algerian black olive cultivars: Effect of dry salting process 1 Introduction 2 Materials and methods 2.1 Olive samples 2.2 Processing of olive samples 2.3 Extract preparation 2.4 Analysis of phenolic compounds 2.4.1 Total phenolic compounds 2.4.2 Total ortho-diphenol content 2.4.3 HPLC analysis of ortho-diphenols 2.5 Antioxidant activity 2.5.1 Reducing power 2.5.2 DPPH free radical scavenging activity 2.5.3 Ferrous-chelating activity 2.6 Statistical analysis 3 Results and discussion 3.1 Total phenolic compounds and o-diphenols 3.2 HPLC identification of o-diphenols 3.3 Antioxidant activity 3.3.1 Reducing power 3.3.2 DPPH free radical-scavenging activity 3.3.3 Ferrous chelating activity 3.3.4 Relationship phenolic contents–antioxidant Activity 4 Conclusion Acknowledgements References