The Occurrence and Bioactivity of Polyphenols in Tunisian Olive Products and by-Products: A Review

Abstract

Abstract:  Polyphenols have become a subject of intense research because of their perceived beneficial effects on health due to their anticarcinogenic, antiatherogenic, anti-inflammatory, and antimicrobial activities. It is well known that olives and their derivatives are rich in phenolic substances with pharmaceutical properties, some of which exert important antioxidant effects. The characterization and quantification of their polyphenol composition is one of the first steps to be taken in any evaluation of the putative contribution of the olive to human health. This review is concerned with polyphenols in Tunisian olive (Olea europaea L.) products (fruit and oil) and some by-products (leaves and olive-mill wastewater) with an emphasis on the analytical methods used, as well as the biological activities described in recent years.

Introduction

Olive-tree cultivation in the Mediterranean area goes back to ancient times. The Romans spread olive cultivation throughout the entire Mediterranean basin. This long-lived tree forms an integral part of the economy and culture of the inhabitants of this basin and also determines its rural landscape (Loumou and Giourga 2003).

Tunisia currently has about 66 million olive trees, covering 1685000 hectares, which represents a 3rd of its available agricultural land. Olive trees are cultivated in widely varied climatic conditions, thus from north to south they are situated as follows: 15% in the north, 66% in the center, and 19% in the south. Olive trees are mostly planted in monoculture and sometimes intercalated with other fruit trees (Larby and Athanasios 2009).

The economic importance of the sector is reflected through its contribution of up to 44% of Tunisian agricultural exports. Tunisia occupies the second position in the world after the European Union in terms of olive-oil exportation (IOOC 2009). During the last decade, olive-oil production reached an average of about 150 thousand tons. The quantity exported represented more than 70% of the total production of olive oil and has achieved close to 111 thousand tons (IOOC 2009).

Besides its economic importance, the olive sector (olive growing and the olive-oil industry) provides a livelihood, directly or indirectly, for over 1 million people and generates 34 million workdays a year, equivalent to over 20% of the employment in agriculture (IOOC). In addition to this important social role, the olive tree contributes amply to the valorization of the less favored areas (marginal regions) in the center and the south, and therefore helps to ensure the stability of rural populations as well as the preservation of the soil because it is a crop that can be grown in difficult conditions.

Virgin olive oil (VOO), the main olive product, which is extracted from olive fruit by mechanical means, is appreciated throughout the world by consumers attentive to both the healthy and nutritional aspects of food. Health-promoting effects may be attributed to the antioxidant effect of the phenolic compounds present in olives and olive oil, and their pharmacological actions have often been reported in the literature (Tuck and Hayball 2002; Ruano and others 2005).

Phenolic compounds or polyphenols are one of the most important groups of compounds occurring in plants, where they are widely distributed, comprising at least 8000 different known structures (Bravo 1998).

Due to the importance of polyphenols in food, this article reviews the polyphenols occurring in Tunisian olive products (olive fruit and olive oil) and some by-products (olive leaves and olive-mill wastewater [OMWW]) together with the various methods used for their analysis and their bioactive properties, in the hope of opening up perspectives for further research.

Olive Products

Olive fruit

Olives attain their maximum weight 8 mo after the flowering period. This is followed by physiological modifications and changes in fruit color with the appearance of the purplish black fruit indicating the end of its morphological development (Amiot and others 1986). The olive ripeness index (RI) can be determined according to the method developed by the Agronomic Station of Jaén (Uceda and Hermoso 1998) based on the evaluation of olive skin and pulp colors. RI values range from 0 (100% intensely green skin) to 7 (100% purple flesh and black skin).

Olives are rarely consumed directly as a natural fruit due to their extreme bitterness but are used instead for the extraction of oil and to a lesser degree as table olives (after curing).

Traditionally, polyphenols are extracted from Tunisian olives by solid–liquid solvent extraction using methanol (Ben Othman and others 2009), ethyl acetate (Fki and others 2005), or mixtures of methanol with water (80 : 20 v/v) (Bouaziz and others 2004; Fki and others 2005).

In a typical extraction procedure, a volume of extractant is added to the olive paste, and this is left to stand overnight under stirring at room temperature. Subsequently, the solution is filtered using GF/F filter paper, and then the extract is washed with hexane in a separatory funnel. The extract is finally concentrated in a vacuum.

Spectrophotometric determination of total phenols For a simple and a rapid quantification of total phenols in olives, the traditional spectrophotometric method based on the Folin-Ciocalteu assay at 727 nm was used. Bouaziz and others (2004) studied the total phenol content in the most important Tunisian olive cultivar, the Chemlali, and studied its variation during ripening. They found that the total phenol concentration of olive extracts increased from 6 to 16 g as pyrogallol equivalents per kilogram fresh weight during ripening. The highest amount was recorded in black olives from the harvest at the end of October to the end of February. Later, Ben Othman and others (2009) studied the effect of ripening (green, varicolored, and black olives) on Chétoui olive flesh (the second most important cultivar in Tunisia) total phenol content at 760 nm. The results showed that green olives have the highest phenolic content of 2.558 g in the form of gallic acid per 100 g dry weight, followed by varicolored and black olives, which had phenolic contents of 2.233 and 1.760 g/100 g dry weight, respectively. The evolution of total phenol content in olive flesh runs inversely to that obtained for the whole olive by Bouaziz and others (2004).

To test the effect of fermentation on olive phenols, Ben Othman and others (2009) used green, varicolored, and black olives of the Chétoui variety. Olives were placed in 8% w/v NaCl brine for 67 days and some were left to undergo spontaneous fermentation, while others were fermented with a selected strain of Lactobacillus plantarum. They observed that the evolution of total phenols was the same in both the olives fermented spontaneously and those fermented with L. plantarum. A significant decrease was observed after 9 days’ fermentation, which continued until day 23, after which the phenolic contents remained stable. Nevertheless, total phenolic reductions for the green, varicolored, and black olives were lower in the controlled than the spontaneous fermentation. This might be explained by the development of a biofilm that could possibly have acted as a barrier for phenolic diffusion (Ben Othman and others 2009).

The popularity of this colorimetric assay can be attributed mainly to its simplicity and speed of analysis (Carrasco-Pancorbo and others 2005). The method is a conventional one, however, since any reducing substance may result in interference, besides which, the response of each phenol to the oxidizing agent is different (Boskou 2009). Furthermore, the method does not distinguish between individual compounds with different molar masses and structures. The need for profiling and identifying individual phenolic compounds requires the replacement of traditional methods by high-performance chromatographic analyses.

Chromatographic determination of the phenolic compounds During ripening, several metabolic processes occur in olives that can lead to a variation of the profiles of some components. Thus, studying the changes in phenolic profile and contents during fruit ripening is of a great interest. Some Tunisian studies have dealt with this subject, focusing mainly upon the Chemlali and Chetoui varieties. Table 1 shows the different analytical methods that provided the highest number of identified phenolic compounds in Tunisian olives.

Table 1–.  Identified phenolic compounds in olive products and the analytical methods used.

Source		Olive fruit		Olive oil
^aIdentified in green and black olive aqueous methanol extracts; ^bidentified in black olive aqueous methanol extract; ^cfor monomer identification; ^dfor flavonoid identification.
Extraction method		SLE		SPE		LLE
		Me-OH : H2O (80 : 20), ethyl acetate	Me-OH : H2O (80 : 20)	diol-bonded phase cartridge		Me-OH : H2O 80 : 40 (v/v)	Me-OH : H2O 60 : 40 (v/v)
Chromatographic technique		HPLC	HPLC	RP-HPLC	HPLC	HPLC	HPLC
	Column	C-18 (4.6 × 250 mm) Shim-pack VP-ODS Lichrosphere 100 RP-18(4 × 250 mm)	C-18 (4.6 × 250 mm) Shim-pack VP-ODS^b C-8 (4.6 × 250 mm) Shim-pack CLC^d	Lichrosphere 100 RP-18, 5 μm (250 × 4 mm)	Phenomenex C18, 5 μm (250 × 3.0 mm)	Lichrosphere 100 RP 18.5 μm (250 × 4 mm)	Zorbax C18 (4.6 × 150 mm, 1.8 μm)
	Mobile phases	0.1% phosphoric acid in water (A) and 70% acetonitrile in water (B)	0.1% phosphoric acid in water (A) and 70% acetonitrile in water (B)	water/phosphoric acid (99.5 : 0.5, v/v) (A) and methanol/acetonitrile (50 : 50, v/v) (B)	0.5% acetic acid in water (A)and acetonitrile (B)	0.2% acetic acid in water (A) and methanol (B)	0.5% acetic acid in water (A) and acetonitrile (B)
	Detector	UV and MS (APCI)	UV (280, 335 nm)	UV (280, 335 nm)	UV (280, 335 nm) MS (ESI-TOF)	UV (278 nm)	UV (240, 280 nm) and MS (ESI-TOF)
Identified compounds		Ol^a, HyTy^a, Ty^a lueolin 7-o-glucoside^a luteolin 7-o-rutinoside^a, quercetin 3-o-glucoside^a, rutin^a, apigenin 7-o-rutinoside^a, chrysoeriol 7-o-glucoside^a, luteolin 4-o-glucoside^a, quercetin^b, luteolin^b, apigenin^b, and chrysoeriol^b	HyTy, Ty, hydroxybenzoic acid, vanillic acid, caffeic acid, coumaric acid, vanillin, ferulic acid, Ol, rutin, quercetin 3-arabino-glucoside, luteolin 7-o-glucoside, quercetin, luteolin, and apigenin	HyTy, Ty, vanillic acid -coumaric acid, HyTy acetate, dialdehydic form of Ol-agl, tyrosol acetate, isomer derivated, dialdehydic form of lig-agl, Pin, Ac-Pin, vanillin aldehydic form of Ol-agl, aldehydic form of lig-agl, ferulic acid, luteolin, and apigenin	HyTy, Ty, HyTy-Ac, EA, D-Ol-agl, Ac-Pin, Ol agl, and lig agl	HyTy, Tyr, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, dialdehydic form of Ol-Agl, o-Coumaric acid, and Ol-Agl	HyTy, Tyr, HyTy-Ac, D-Ol agl, D-Lig-agl, Ol-agl, H- D-Ol agl, methyl- D-Ol agl, 10-H-Ol agl, Lig-agl, vanillic acid, o-coumaric acid, ferulic acid, taxifolin, EA, H-EA, Pin, Ac-Pin, -Pin, syringaresinol, sinapinic acid, luteolin, and apigenin
Olive cultivars		Chemlali	Chemlali	Chetoui, Chemlali, Sayali, Oueslati, Gerboui, Chemchali, and Zalmati	Chetoui	Chetoui and Chemlali	Chetoui, Chemlali, El Hor, Jarboui, Chemchali, and Oueslati
References		(Fki and others 2005)	(Bouaziz and others 2004)	(Abaza and others 2005)	(Ben Youssef and others 2009)	(Guerfel and others 2009)	(Taamalli and others 2010)

Bouaziz and others (2004) characterized the phenolic compounds in the Chemlali olive in order to examine their profile during ripening (from July 1 to February 20). Using reversed-phase high-performance liquid chromatography with UV detection (RP-HPLC-UV), they identified 15 phenolic compounds: hydroxytyrosol (HyTy), tyrosol (Ty), p-hydroxybenzoic acid, vanillic acid, caffeic acid, coumaric acid, vanillin, ferulic acid, oleuropein (Ol), rutin, quercetin 3-arabino-glucoside, luteolin 7-o-glucoside, quercetin, luteolin, and apigenin (Table 1). Quantification of phenolic compounds in Chemlali olives during ripening showed that the highest quantity was recorded for Ol, which reached a maximum value of 6.5 g/kg fresh weight for the sample harvested on August 30 (green fruit). The Ol concentration varied greatly during sampling, however, increasing from 2.5 g/kg until reaching a maximum value by the end of July. After that, its concentrations decreased until reaching a minimum value of 1.5 g/kg during the last stage of ripening. Similar results have been reported in other studies in which the Ol concentration was higher in green Chemlali olives (89.65% of dry weight extract) (Fki and others 2005) and in green Chétoui olive flesh (266 mg gallic acid/100g dry weight) (Ben Othman and others 2009).

The HyTy and Ty concentrations in Chemlali olives increased during ripening and reached their maximum values at the last ripeness stage (Bouaziz and others 2004). The evolution of these two compounds is opposite to that found in olive flesh by Ben Othman and others (2009). Ferulic acid remained relatively constant; p-coumaric acid showed a weak variation and decreased in the last phase of ripening. Caffeic acid, p-hydroxybenzoic acid, vanillic acid, and vanillin were present in low concentrations. With regard to flavonoids, luteolin-7-o-glucoside, quercetin-3-arbino-glycoside, and rutin increased during olive ripening until reaching maximum quantities in mid-September, November 4, and mid-November, respectively. After these dates, the quantities of each compound decreased. Quercetin concentrations showed very little change throughout ripening. Apigenin remained substantially unchanged and quite low throughout all the ripening stages.

In a further study, Fki and others (2005) used a combination of HPLC and atmospheric-pressure chemical-ionization mass spectrometry (HPLC-APCI-MS) in positive ion mode. In addition to the flavonoids reported previously by Bouaziz and others (2004), they identified six flavonoids in Chemlali olive extract: luteolin 7-o-rutinoside, quercetin 3-o-glucoside, apigenin-7-o-rutinoside, chrysoeriol-7-o-glucoside, luteolin-4-o-glucoside, and chrysoeriol (Table 1). They also found that some of these flavonoids, quercetin, luteolin, apigenin, and chrysoeriol, were only present in the aqueous methanol extract from black olives.

Changes in phenolic compounds during olive processing (fermentation) were studied by Ben Othman and others (2009). These authors studied olive-flesh phenolic compounds during spontaneous and controlled fermentations of Chétoui olives at 3 stages of ripeness (green, varicolored, and black olives) using the HPLC-UV technique. After fermentation, changes in the quantity of phenolic compounds were observed; phenolic content in flesh increased after the fermentation of varicolored and black olives, especially in the controlled fermentation (from 384 and 311 to 621 and 510 mg gallic acid/100 g dry weight, respectively). In contrast, the phenolic content decreased in green olives from 652 to 460 mg gallic acid/100 g dry weight and to 380 mg/100 g dry weight in spontaneous and controlled fermentations, respectively. There was a decrease in the concentrations of protocatechuic acid, ferulic acid, and oleuropein, while HyTy concentration increased after fermentation, due to the acid and enzymatic hydrolysis of oleuropein. The concentrations of gallic, p-hydroxyphenylacetic, vanillic, and benzoic acids also decreased after the fermentation of green olives, although their concentrations increased for varicolored and black olives.

BioactivityFki and others (2005) studied the lipid-lowering effect and the antioxidative activities of the Tunisian Chemlali green and black olive phenolic extracts (Table 2). Wistar rats fed on a standard laboratory diet or a cholesterol-rich diet for 16 wk were used. The results showed that the administration of aqueous methanol and ethyl acetate extracts of green olives and ethyl acetate extract of black olives significantly lowered the serum levels of total cholesterol and low-density lipoprotein cholesterol while increasing the serum level of high-density lipoprotein cholesterol. Furthermore, the malondialdehyde content in the liver, heart, and kidney decreased significantly after oral administration of green and black olive extracts compared to those of rats fed on a cholesterol-rich diet. In addition, olive extracts increased catalase and superoxide dismutase activities in the liver (Table 2). The hypocholesterolemic and antioxidative effects of aqueous methanol and ethyl acetate extracts of green olives and ethyl acetate extract of black olives could be related to their HyTy- and Ol-rich contents (Fki and others 2005).

Table 2–.  Bioactive effects of olive products and by-products.

Bioactive effect	Cell/organ	Extract type and concentration range tested	Effective concentration/extract	Source and variety	Reference
Hypocholesterolemy	Serum of Wistar rats fed a cholesterol-rich diet	Methanol or ethyl acetate extracts of green and black olives (5 mg/kg of body weight for each extract)	Ethyl acetate green olive extract, methanol green olive extract, ethyl acetate black olive extract	Olives: Chemlali	Fki and others 2005
Increase of antioxidant enzyme activity (CAT, SOD)	Liver of Wistar rats fed a cholesterol-rich diet	Methanol or ethyl acetate extracts of green and black olives (5 mg/kg of body weight for each extract)	Ethyl acetate green olive extract, methanol green olive extract, ethyl acetate black olive extract	Olives: Chemlali	Fki and others 2005
Lipid peroxidation reduction	Liver, heart, and kidneys of Wistar rats fed a cholesterol-rich diet	Methanol or ethyl acetate extracts of green and black olives (5 mg/kg of body weight for each extract)	Ethyl acetate green olive extract, methanol green olive extract, ethyl acetate black olive extract	Olives: Chemlali	Fki and others 2005
Antiproliferation	Promyelocytic leukemia HL-60 human cell	Ethanol olive-leaf extracts at 1/100–1/10000 dilutions	Chemlali extract at 1/100 dilution	olive leaves: Chemchali, Chemlali, Chetoui, Gerboui, Sayali, Zarrazi, and Zalmati	Abaza and others 2007
Cell apoptosis	Promyelocytic leukemia HL-60 human cell	Ethanol olive-leaf extract at 1/100 dilution	Chemchali, Chemlali, and Zalmati extracts at 1/100 dilution	Olive leaves: Chemchali, Chemlali, Chetoui, Gerboui, Sayali, Zarrazi, and Zalmati	Abaza and others 2007
Cell differentiation	Promyelocytic leukemia HL-60 human cell	Ethanol olive-leaf extract at 1/100 dilution	Gerboui extract	Olive leaves: Chemchali, Chemlali, Chetoui, Gerboui, Sayali, Zarrazi, and Zalmati	Abaza and others 2007
Inhibition of β-hexosaminidase release at antigen–antibody binding	Rat basophilic leukemia (RBL-2H3) cells	Olive-oil emulsions at 1/100–1/10000 dilutions	Sayali at 1/10000 dilution	Olive-oil emulsions: Chemlali, Chemchali, Chetoui, Sayali, Zarrazi, Zalmati, and Gerboui	Yamada and others 2009
Inhibition of β-hexosaminidase release at antibody-receptor binding	Rat basophilic leukemia (RBL-2H3) cells	Olive-oil emulsions at 1/100–1/10000 dilutions	Zarrazi at 1/100 dilution	Olive-oil emulsions: Chemlali, Chemchali, Chetoui, Sayali, Zarrazi, Zalmati, and Gerboui	Yamada and others 2009
Inhibition of histamine release	Human basophilic (KU812) cells	Olive-oil emulsions at 1/100–1/10000 dilutions	Zarrazi at 1/100 dilution	Olive-oil emulsions: Sayali and Zarrazi	Yamada and others 2009
Hypolipidimy (TC, LDL-C, and TG)	Serum of rats fed on a cholesterol-rich diet	Ol-, ol agly-, and HyTy-rich extracts 3 mg/kg of body weight	Tested extracts	Olive leaves: Chemlali	Jemai and others 2008
Increase of antioxidant enzyme activities (CAT and SOD)	Liver of rats fed on a cholesterol-rich diet	Ol-, ol agly-, and HyTy-rich extracts 3 mg/kg of body weight	Tested extracts	Olive leaves: Chemlali	Jemai and others 2008
Serum antioxidant potential	Serum of rats fed on a cholesterol-rich diet	Ol-, ol agly-, and HyTy-rich extracts 3 mg/kg of body weight	Tested extracts	Olive leaves: Chemlali	Jemai and others 2008
Lipid peroxidation reduction (TEBARS levels)	Liver, heart, and aorta homogenates of rats-rich diet	Ol-, ol agly-, and HyTy-rich extracts 3 mg/kg of body weight	Tested extracts	Olive leaves: Chemlali	Jemai and others 2008
Effect on histopathology	Liver, heart, and aorta tissues of rats fed on a cholesterol-rich diet	Ol-, ol agly-, and HyTy-rich extracts 3 mg/kg of body weight	Tested extracts	Olive leaves: Chemlali	Jemai and others 2008

Olive oil

In the kitchens of consumers, olive oil is often the fat of choice for health conscious people looking to enjoy the benefits of the Mediterranean diet. It is of great interest for its healthy virtues. Its chemical composition is principally triacylglycerols, which account for more than 98% of its total weight. Minor components amount to about 2% of the total weight and include, among others, aliphatic and triterpenic alcohols, sterols, hydrocarbons, volatile compounds, and several antioxidants (Servili and Montedoro 2002). Phenolic compounds represent one of these families of antioxidants present in olive oil.

Two basic extraction techniques, liquid–liquid extraction (LLE) and solid-phase extraction (SPE), have been described for isolating polyphenols from Tunisian olive-oil samples (Abaza and others 2005; Ben Temime and others 2006; Baccouri and others 2007; Krichène and others 2007; Mahjoub-Haddada and others 2007; Ben Youssef and others 2009; Guerfel and others 2009; Taamalli and others 2010; Issaoui and others 2010). Table 1 shows the different analytical methods that have identified the highest number of phenolic compounds in Tunisian olive oil.

As far as the LLE is concerned, the phenolic fraction of olive oil is isolated by the extraction of an oil solution in hexane with several portions of methanol/water (at different ratios), followed by solvent evaporation of the aqueous extract. Some authors have used methanol/water at a ratio of 60 : 40 v/v (Ben Temime and others 2006; Mahjoub-Haddada and others 2007; Baccouri and others 2007; Taamalli and others 2010) or a ratio of 80 : 20 v/v (Guerfel and others 2009; Issaoui and others 2010).

With regards to the SPE, the polyphenols of Tunisian olive-oil samples have been studied using a diol-bonded phase SPE cartridge (Abaza and others 2005; Krichène and others 2007; Ben Youssef and others 2009). The experimental approach was as follows: a sample of filtered olive oil was dissolved in 6 mL of n-hexane. A diol-bonded phase cartridge was placed in a vacuum elution apparatus and conditioned by the consecutive volumes of methanol and hexane. The vacuum was then released to prevent drying of the column. The oil solution was applied to the column and the solvent was pulled through, leaving the sample and the standard on the solid phase. The sample container was washed with hexane, which was run out of the cartridge. The sample container was washed again with hexane/ethyl acetate, which were run out of the cartridge and discarded. Finally, the column was eluted with methanol and the solvent was evaporated in a rotary evaporator at room temperature and low speed under a vacuum until dry.

Spectrophotometric determination of total phenols The colorimetric assay based on the reaction of Folin–Ciocalteu reagent was used for the determination of total phenols. Total o-diphenols were determined separately with a solution of sodium molybdate in ethanol/water (50 : 50 v/v).

These colorimetric assays were used to evaluate the effects of genotype, geographic location, ripening, and irrigation on the total phenol and o-diphenol contents of VOO.

Zarrouk and others (2008) analyzed the variation in total phenol contents among several olive-oil varieties grown in southern Tunisia. The quantities of total phenols and o-diphenols showed significant differences among cultivars. Zalmati oil contained the highest total phenol concentration (507 mg of caffeic acid/kg of oil), whereas Jemri Ben Guerdane oil recorded the lowest (364 mg/kg). The highest o-diphenol content was observed in Chemlali Zarzis oil (213 mg/kg), followed by “Jemri Ben Guerdane” (about 199 mg/kg), while “Zalmati” showed the lowest value (188 mg/kg). Nevertheless, when exploring 10 different farms in the north of the country where the Chétoui variety is cultivated, Ben Temime and others (2006) found that Chétoui olive oil from the region of Amdoun had very high levels of total phenols and o-diphenols, which exceeded 740 and 280 mg of caffeic acid/kg of oil, respectively.

Mahjoub-Haddada and others (2007) studied total phenol contents in VOO from Tunisian olive varieties cultivated in the north of the country. They found that oils from Chétoui showed higher values for phenols and o-diphenols (321.68 and 33.17 mg of caffeic acid/kg of oil, respectively) than the other varieties from the same area studied.

Baccouri and others (2007) evaluated the influence of an irrigation regime on the total phenols and o-diphenols in Chétoui VOO at 3 different stages of ripeness. They found that total phenol quantities were significantly affected by the irrigation regime. In fact, oils obtained from irrigated trees had lower levels of total phenols (which did not exceed 294 mg of gallic acid/kg of oil) than nonirrigated ones (which reached 852 mg/kg). In the rain-fed Chétoui variety, the total phenol contents increased to reach a maximum level at stage II of fruit ripening, then the total phenol content decreased and no evident losses in o-diphenol content were observed. The total phenol and o-diphenol contents decreased with ripeness, however, when the Chétoui cultivar was under irrigation.

Similar behavior was found by Ben Youssef and others (2009). These authors observed that the quantities of total phenol increased progressively during the ripening of Chétoui olives until they reached a maximum value (1043 mg of caffeic acid/kg of oil) at a ripeness index of between 2 and 3.5, after which it decreased to a minimum of 483 mg/kg.

Chromatographic determination of the phenolic compounds in VOO HPLC has been the analytical technique most used for characterizing polyphenolic compounds. As can be seen in Table 1, the phenolic profile of olive oil is different from that of the fruit, where the glycosidic forms are predominant. In addition, the analytical method used has an influence on the determination of the phenolic compounds.

Using RP-HPLC-UV, Abaza and others (2005) studied the phenolic composition of seven Tunisian olive-oil varieties. They identified the following phenolic compounds: HyTy, Ty, p-coumaric acid, ferulic acid, vanillic acid, vanillin, HyTy-acetate, Ty-acetate, dialdehydic forms of ligostroside and Ol aglycones, isomerderivated, aldehydic forms of ligostroside and oleuropein aglycones, apigenin, luteolin, pinoresinol (Pin), and acetoxypinoresinol (Ac-Pin) (Table 1).

Two of these seven varieties (Chétoui and Chemlali) were studied by Baccouri and others (2008) and later by Guerfel and others (2009). Baccouri and others (2008) used HPLC coupled to a UV-Vis detector and a mass-spectrometer detector (MS) equipped with an electrospray ionization (ESI) interface operating in positive mode. They identified phenolic compounds in these two varieties that had not previously been identified by Abaza and others (2005), such as vanillic and o-coumaric acids. In 2009, Guerfel and others analyzed the phenolic composition of VOO from the same varieties (Chétoui and Chemlali) using the HPLC-UV system. They identified three additional phenolic acids: ferulic, syringic, and caffeic acids (Table 1). By using an HPLC system coupled to a time-of-flight mass spectrometer (TOF-MS) equipped with an ESI operating in negative ion mode, Taamalli and others (2010) were able to identify other phenolic compounds that had not previously been reported in Chétoui olive oil: elenolic acid (EA), hydroxy-elenolic acid (H-EA), hydroxy-D-oleuropein aglycon (H-D-Ol agl), methyl-D-Ol agl, 10-H-Ol agl, syringaresinol, hydroxy-Pin (H-Pin), and sinapinic acid (Table 1).

Studies showed that the quantities of the phenolic compounds differ between olive-oil varieties (Abaza and others 2005; Krichène and others 2007; Baccouri and others 2008; Ben Youssef and others 2009; Taamalli and others 2010). Abaza and others (2005) found that the prevalent compounds were the aldehydic form of ligstroside and oleuropein aglycones and their dialdehydic forms with levels reaching 124.43, 211.39, 405.53, and 66.79 mg/kg, respectively. The major simple phenol alcohols were HyTy and Ty, which varied from 0.81 to 23.73 mg/kg and from 2.66 to 33.17mg/kg, respectively. The contents of the two main flavonoids identified in the olive oils studied luteolin and apigenin were very low and did not exceed 6 mg/kg. As far as the lignans are concerned, Ac-Pin was the most abundant and exceeded 13 mg/kg. Among the oils analyzed, the Chétoui variety had the highest phenolic content (925.7 ± 24.3 mg/kg).

In a study by Taamalli and others (2010), the Chemchali and El Hor varieties showed interesting levels of phenolic compounds compared to the Chetoui variety. The polyphenols reached 3564 mg/kg.

Baccouri and others (2008) studied differences in the phenolic composition of the two main monovariety Tunisian VOOs, Chétoui and Chemlali at 5 different stages of ripeness. The Chétoui cultivar was also tested in an irrigation regime against a rain-fed control. The concentration of phenolic compounds gradually increased during ripening until reaching a maximum value at the ‘‘reddish” and ‘‘black” pigmentation stage (RI between 3 and 4), after which it decreased. The analysis showed that olive-oil samples contain low quantities of phenyl acids and phenyl alcohols and high quantities of secoiridoid derivatives such as oleuropein aglycon Ol agl, lig agl, and D-Ol agl. The secoiridoid derivative levels behaved similarly during ripening in both varieties: they increased to reach a maximum level recorded at an RI ranging between 3.5 and 4.1, before rapidly decreasing again. Ol agl was the main secoiridoid derivative identified in all the VOOs analyzed. The evolution of its levels was similar to the secoiridoid derivative content throughout the ripening process. D-Ol agl showed an important variation in Chemlali oils compared to those in the Chetoui variety, its levels decreasing progressively from 12.6 mg of 3,4-dihydroxyphenylacetic acid/kg of oil down to 1.8 mg/kg. Lig agl was present at lower levels than other secoiridoids. In Chétoui oils, its levels decreased slightly as ripening progressed from 10.9 to 6.4 mg/kg, whereas in the Chemlali samples, the content of this compound increased to reach a maximum value of 19.75 mg/kg and then decreased to 3.56 mg/kg. In Chétoui VOOs, the levels of simple phenols (sum of Ty, HyTy, vanillic, and o-coumaric acids) increased as ripening progressed, whereas this behavior was not observed in Chemlali samples.

Phenolic compound levels in Chétoui oils were significantly affected by the irrigation regime applied to the olive trees during ripening (Baccouri and others 2008). Chétoui oils obtained from unirrigated trees had higher quantities of secoiridoid derivatives, ranging from 263.8 to 510.1 mg/kg. In fact, in samples obtained under the irrigation system, rather than rain-fed conditions, secoiridoid-derivative contents decreased slightly as ripening progressed. The simple phenol levels were also affected by the irrigation regime throughout ripening; they increased markedly at the three first ripeness indexes to reach a maximum quantity of 109.4 mg/kg, and then decreased to 48.3 mg/kg. The authors concluded that the early harvest date for the Chétoui cultivar (RI between 3 and 4) gave the best results, while, for the Chemlali cultivar, the data clearly showed higher values of phenolic contents between the 2nd and the 3rd harvest dates (RI between 2.8 and 4.5).

To determine the effect of geographic location on the quantities of phenolic compounds identified, Guerfel and others (2009) analyzed the Chétoui and Chemlali olive-oil varieties cultivated in different locations. They found that the Chétoui variety was subject to greater alteration than Chemlali according to geographic location. Chétoui cultivated in its traditional area produced oil richer in HyTy, Ty, and the dialdehydic forms of Ol agl and Ol agl, which reached 17.82, 23.93, 42.86, and 744.9 mg/kg, respectively. Other simple phenols such as vanillic acid, o-coumaric acid, caffeic acid, syringic acid, and ferulic acid were found in low concentrations.

An investigation into the behavior of the phenolic composition of VOO under medium-temperature-accelerated storage conditions was undertaken by Krichen and others (2010). The changes undergone by phenolic compounds were studied under medium temperature (50 °C) accelerated oxidation conditions over a storage period of 8 mo. Different oxygen availability (open and closed bottles) and four different monovariety Tunisian VOOs were used. The results showed that the content of both oleuropein-derived aglycons (aldehydic and dialdehydic forms of Ol-agl) and ligstroside-derived aglycons (aldehydic and dialdehydic forms of Lig-agl) were rapidly reduced under the average temperature (50 °C) accelerated storage conditions of the assay. The initial degradation rate observed in the secoiridoid compounds was quite significant, even in the closed bottle samples, although 2–4 times lower than in open bottles. As a consequence, the residual quantity of complex phenolic compounds in the closed bottle samples after the 8 months’ accelerated storage at 50 °C was significantly higher than in open-bottle samples, in particular when the initial concentration of oleuropein- or ligstroside-derived compounds was high. The behavior of both HyTy and Ty in closed-bottle stored samples during the 8 mo assay was very similar, whereas their behavior was different in open-bottle stored samples, in which a greater stability of Ty could be detected, showing that the very different behaviors observed between these two compounds was mainly due to the antioxidant role of HyTy.

Bioactivity Tunisian olive oils were investigated for their antiallergic property. To this end, Yamada and others (2009) studied 5 olive varieties grown in various regions of Tunisia. They found that among the varieties studied, Sayali olive oil presented the most potent inhibitory effect on β-hexosaminidase release by the Immunoglobulin E antibody-sensitized, Bovine serum albumin antigen-simulated RBL-2H3 cells at the antibody–antigen binding stage. The results of the experiment showed that the antiallergic effect of olive oils at this binding stage may depend upon their flavone content. At the antibody-receptor stage, among the varieties studied, Zarrazi olive oil showed a higher inhibitory effect on β-hexosaminidase release from RLB-2H3 cells. They investigated the effect of olive oil samples on histamine release and cytokine production by activated human basophilic (KU812) cells. Different dilutions of Zarrazi olive oil inhibited histamine release from A23187 and phorbol 12-myristate 13-acetate (PMA)-stimulated KU812 cells in a dose-dependent way. Therefore, this study showed that the consumption of Tunisian olive oils may be beneficial for the prevention and even treatment of various types of allergy (Table 2).

Olive By-Products

Both the cultivation of olive trees and olive oil extraction generate substantial quantities of products generally known as “olive by-products.” The different by-products considered in this review are defined as follows: olive leaves and OMWW. Table 3 shows the analytical methods that have identified the highest number of phenolic compounds in Tunisian olive by-products.

Table 3–.  Phenolic compounds identified in olive by-products and the analytical methods used.

Source		Olive leaf	OMWW
^aFor monomer identification; ^bfor flavonoid identification.
Extraction method		SLE	Continuous counter current extraction
		Methanol : water (80 : 20)	Ethyl acetate
Chromatographic		HPLC	RP-HPLC
Technique
	Column	C-18 (4.6 × 250 mm) Shim-pack VP-ODS^a Lichrosphere 100 RP-18 (4 × 250 mm)^b	4.6 × 250 mm (Shim-pack VP-ODS)
	Mobile phases for HPLC	0.1% phosphoric acid in water (A) and 70% acetonitrile in water (B)^a 2% acetic acid in water (A) and methanol, acetic acid, and water (18 : 1 : 1) (B)^b	0.1% phosphoric acid in water (A) and 70% acetonitrile in water (B)
	Detection system	UV (at 280 nm) ^a	UV (at 280 nm)
		photodiode array (APCI)-MS^b
Identified compounds		ol, luteolin 7-o-glucoside, luteolin 7-o-rutinoside, apigenin 7-o-glucoside, rutin, luteolin, and apigenin	HyTy, 3,4-dihydroxyphenylacetic acid, HyTy-4-β-glucoside, p-coumaric acid, ferulic acid, tyrosol, and Ol
Olive cultivars		Chemlali (Sfax)	Chemlali
References		(Bouaziz & Sayadi, 2005)	(Feki and others 2006)

Olive leaves

Large amounts of olive leaves are one of the by-products of olive farming; they accumulate during the pruning of the olive trees and are also account for up to 10% of the total weight of the olives at olive-oil mills (Tabera and others 2004).

Two different solvent mixtures were used for the extraction of polyphenols from olive leaves: methanol/water (Bouaziz and Sayadi 2005; Jemai and others 2008) and ethanol 70% (Abaza and others 2007).

Spectrophotometric determination of total phenols Total phenols in olive leaves were also determined using the Folin–Ciocalteu reaction. Boudhrioua and others (2009) showed a variation among the studied Tunisian cultivars from 1.4 to 2.32 mg as caffeic acid/100g of dry matter, Chetoui and Zarrazi being the richest ones.

Chromatographic determination of the phenolic compounds in olive leaves Not enough information exists concerning the phenolic composition of Tunisian olive leaves, and studies that have been made have only focused on the major phenolic compounds.

Abaza and others (2007) studied the phenolic composition of some Tunisian olive-leaf extracts by using reversed-phase HPLC-UV. They found that 2 main compounds were present in all the extracts: apigenin-7-o-glycoside and Ol. Their quantities varied among the varieties studied from 1.31 to 2.68 and from 0.91 to 2.81 g as o-coumaric acid/kg dry weight, respectively. The highest quantity of apigenin-7-o-glycoside was found in the Zalmati cultivar, while the Chemchali cultivar presented the highest quantity of Ol. A few minor compounds were present but were not detailed.

With the aim of studying the evolution of the quantity of the major phenolic compounds found in Chemlali olive leaves, Bouaziz and others (2005) used leaves collected from July 2003 to March 2004. Extract analysis was performed by HPLC, which revealed that Ol was always the major compound. This accords with the findings of Jemai and others (2008), who found that the major phenolic compound in Chemlali olive-leaf extract, Ol, reached 4.32 g/100 g dry weight. Bouaziz and others (2005) found low variations in Ol concentration during the whole period of harvest (from 12.4% to 14.2%). Besides Ol, they identified flavones (luteolin 7-o-glucoside, luteolin 7-o-rutinoside, apigenin 7-o-glucoside, luteolin, and apigenin) and one flavonol (rutin) using a combination of HPLC-UV and APCI-MS but did not quantify them (Table 3).

BioactivityAbaza and others (2007) investigated the protective effects of seven olive-leaf extracts of Tunisian olive varieties against human leukemia (Table 2). The extracts showed an antiproliferative effect on HL-60 cells incubated for 48 h and the most potent was the Chemlali extract. The Gerboui extract showed the highest capacity to reduce nitroblue tetrazolium. Apigenin-7-glucoside was mainly responsible for the differentiation of HL-60 cells mediated by Gerboui extract.

Jemai and others (2008) investigated the hypolypidemic and antioxidant activities of Ol and its hydrolysis-derivative-rich extracts in rats fed on a cholesterol-rich diet. The results showed that the administration of polyphenol-rich olive leaf extracts significantly lowered the serum levels of total cholesterol and triglycerides and low-density lipoprotein cholesterol while at the same time increasing the serum level of high-density lipoprotein cholesterol (HDL-C). In addition, these extracts lowered the content of thiobarbituric acid reactive substances in the liver, heart, kidneys, and aorta compared with those of rats fed on a cholesterol-rich diet. In addition, they increased the serum antioxidant potential and hepatic superoxide dismutase and catalase activities (Table 2).

OMWW

OMWW is a by-product of the 3-phase process of the extraction of oil from olives. This black wastewater is composed of the olive-fruit vegetation water, the water used for washing and treating the olives and a portion of the pulp and residual oil (Ben Sassi and others 2006).

A continuous counter-current extraction procedure has been reported for the removal of polyphenols from Tunisian OMWW (Fki and others 2005; Feki and others 2006; Khoufi and others 2008). Among several polar solvents such as methyl isobutyl ketone, methyl ethyl ketone, and diethyl ether, ethyl acetate was found to extract broadly the whole OMWW monomeric fraction (Fki and others 2005).

Chromatographic determination of the phenolic compounds in OMWW An RP-HPLC system coupled to a UV detector was used to identify the major phenolic compounds of the OMWW extract (Fki and others 2005). HyTy and Ty were the major compounds in the OMWW extract at concentrations of 1225 and 345 mg/L, respectively; lower concentrations of p- hydroxyphenyl acetic acid, caffeic acid, and p-coumaric acid were present. 3,4-Dihydroxyphenylacetic acid and ferulic acid were also present at the same concentration, 70 mg/L. Protocatechuic acid, vanillic acid, syringic acids, and other compounds were detected but not quantified.

In another study, also using RP-HPLC, Feki and others (2006) studied the storage effect on the phenolic composition of OMWW (Table 3). Fresh and stored Chemlali OMWWs of two harvest periods were used. Two compounds, H-4-β-glucoside and Ol, were not cited by Fki and others (2005). Among the compounds identified, HyTy was the most abundant phenolic monomer in both fresh and stored OMWW. Its concentration increased during storage for both harvest periods, varying over a 5 mo period from 0.98 to 3.5 g/L for OMWW from the harvest period 2004–2005, and from 0.77 to 3.1 g/L after 4 mo storage from the harvest period 2005–2006. In contrast to the evolution of HyTy, the concentration of Ol decreased markedly in the extracts from both harvest periods. This evolution might be put down to some hydrolysis reactions of HyTy derivatives composed of HyTyt units attached to other compounds via ester and/or glucosidic linkages. These hypotheses accord with those of Gómez-Caravaca and others (2011).

Comparison between the Tunisian Olive Phenolic Compounds and Bioactivity and Some Mediterranean Varieties

As we have already mentioned in this review, several phenolic compounds from Tunisian olive tree derivatives (fruit, oil, leaf, and OMWW) exert biological activity. In comparison to the biological activity of other Mediterranean olive derivatives, it is interesting to note that the antiallergic property of olive oils has been studied for the first time in Tunisian olive oils. As far as the bioactive compounds are concerned, Ol, HyTy, Ol agl, apigenin-7-glucoside, and other flavones such as apigenin and luteolin present in Tunisian olive derivatives exhibit different biological activities. It has been reported that Ol and HyTy inhibit the copper-sulphate-induced oxidation of LDL strongly and dose dependently (Visiolli and Galli 1994; Visiolli and others 1995). HyTy has also proved to be effective in a model of oxidative stress induced in intestinal epithelial cells (Manna and others 1997) and in building up plasma antioxidant capacity (Visiolli and others 2001).

Identified in other oils, it has also demonstrated antimicrobial properties (Bisignano and others 1999), antiproliferative effect against human promyelocytic HL60 leukemia cells and human colon cancer lines (Fabiani and others 2006; Gill and others 2005; Fini and others 2008).

On the basis of olive oil being the most important product of the olive tree, we have set out in Table 4 some olive oils produced in the Mediterranean basin together with their phenolic composition and bioactivity. According to the bibliography, the main phenolic compounds identified in Tunisian olive oils have also been reported in other olive oils from Mediterranean countries. As Table 4 shows, not many studies have been undertaken into the bioactivity of Tunisian olive oils and so it would be interesting to investigate other biological activities of Tunisian olive oils as some varieties showed interesting phenolic profiles.

Table 4–.  Comparison of phenolic compounds present in olive oils from some Mediterranean olive varieties together with their bioactivity.

Country	Tunisia	Spain	Italy	Turkey	Greece
Studied cultivars	£Chetoui, Chemlali, Chemchali, Sayali, Zarrazi, Zalmati, and Gerboui	¥Arbequina, Hojiblanca, Cornozuelo, and Manzanilla	EVOOs on current Italian market	^¤Memicik, Erkence, Nizip-yaglik, Gemlik, and Ayvalik,	Lianolia
				*Ayvalik
	¤Chetoui, Chemlali, El Hor, Jarboui, Chemchali, and Oueslati	#Picual, Manzanilla, Cornicabra, and Hojiblanca
Phenolic compounds	£HyTy, Ty, VA, p-coumaric acid, HyTy-ac, dialdehydic form of Ol agl, Ty-ac, isomer derivativated, dialdehydic form of Lig agl, pinoresinol, Ac-pinoresinol, vanillin, aldehydic form of Ol agl, aldehydic form of Lig agl, FA, luteolin, apigenin	¥HyTy, Ty, vanillin, p-coumaric acid, HyTy-ac, EA, Hy-EA, decarboxymethyl Ol agl, H-D-Ol agl, syringaresinol, pinoresinol, decarboxymethyl lig agl, H-D-Lig agl, 10-H-Ol agl, Ol, agl, luteolin, methyl D-Ol agl, Lig agl, apigenin, and methyl Ol agl	^◊HyTy,Ty, Ol agl, and Lig agl	^¤HyTy, 2,3-dihydroxybenzoic acid, Ty, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, VA, CA, vanillin, p-coumaric acid, FA, cinnamic acid, luteolin, and apigenin	HyTy, Ty, VA, Syringic acid, HyTy derivative, Ty derivative, and complex phenolic compounds
	¤HyTy, Tyr, HyTy-Ac, D-Ol Agl, D-Lig-Agl, Ol-Agl, H- D-Ol Agl, methyl- D-Ol Agl, 10- H-Ol Agl, Lig-Agl, vanillic acid, o-coumaricd acid, ferulic acid, taxifolin,EA, H-EA, Pin, Ac-Pin, H-Pin, syringaresinol, sinapinic acid, luteolin, and apigenin
		#HyTy, Ty, HyTy-Ac, HyTy glycol, Lig agl, Ol agl, lignans, and flavones not specified	^2HyTy, Ty, dial dehyd D-Ol agl, dialdehyd D-Lig agl, (+)-1-acetoxypinoresinol, (+)-pinoresinol, Ol agl, and Lig agl	*Hyty, Ty, vanillin, p-coumaric acid, luteolin, apigenin, dialdehyd Ol agl, pinoresinol, aldehyd Ol agl, and aldehyd Lig agl
Bioactivity	£Antiallergy	¥Antibreast cancer	^◊Antioxidative stress
		#Antimicrobial	^2Antioxidant/anticancer
Interesting varieties	£Sayali, Zarrazi	¥ Picual (Cordoba)
References	£Yamada and others 2009	¥Lozano-Sánchez and others 2010	^◊Manna and others 2002	^¤Ocakoglu and others 2009	Tasioula-Margari and Out 2001
	¤Taamalli and others 2010	#Medina and others 2006	^2Owen and others 2000	*Andjelkovic and others 2009

Conclusions

As can be seen from this review, the composition and quantity of phenolic compounds in olive products and by-products are the result of a complex interaction of various factors. From the existing literature concerning the characterization of polyphenols in the Tunisian olive and its derivatives, it is clear that the main focus has been directed toward the two most important varieties: Chétoui and Chemlali. Numerous phenolic compounds have been identified and quantified and some of them show interesting biological activities. Nevertheless, bearing in mind the diversity of Tunisian olive germplasm, a widening of the study to other varieties is called for. Investigation of other olive-tree organs such as vegetative and flowering buds and flowers may also be of interest. Apart from this, research should be undertaken into the role of polyphenolics in the regulation of flower and fruit development in olives.

With regard to extraction procedures, modern technologies such as accelerated solvent extraction, supercritical, or superheated fluid extraction and microwave-assisted solvent extraction, used for accelerating, automating the removal step, and/or manipulating the extract characteristics, are notable for their absence in this field of research. As far as the analytical methods are concerned, the method of choice tends to be HPLC due to its high resolution, high efficiency, high reproducibility, and relatively short analysis time without restrictions on sample volatility. Moreover, HPLC has been coupled to a variety of detectors such as UV and MS.

The biological activities of olive derivatives may have a significant impact on the health population by reducing the development of chronic degenerative disease. Thus, in view of the growing importance of polyphenols, research in this field may provide more information about olive polyphenols and open up new possibilities for practical applications of their bioactivity and for adding value to olive by-products.

Acknowledgments

The authors are grateful to the Tunisian Ministry of Higher Education and Scientific Research for their financial support and to the Spanish Ministry of Education and Science for project AGL2011-29857-C03-02. They also thank the Andalusian Regional Government Council of Innovation and Science for the excellence projects P10-FQM-6563 and P11-CTS-7625 and the University of Granada for the GREIB project GREIB.PYR-2011-02. The authors also thank their colleague A.L. Tate for revising their English text.

Nomenclature

• Ac-Pin
    Acetoxy-pinoresinol
• APCI
    Atmospheric-pressure chemical ionization
• CAT
    Catalase
• D-Lig agl
    Decarboxylated ligstroside aglycon
• EA
    Elenolic acid
• ESI
    Electrospray ionisation
• HDL-C
    High-density lipoprotein cholesterol
• H-D-Ol agl
    Hydroxy-decarboxymethyl-oleuropein aglycon
• H-EA
    Hydroxy-elenolic acid
• H-Pin
    Hydroxy-pinoresinol
• HPLC
    High-performance liquid chromatography
• HyTy
    Hydroxytyrosol
• HyTy-Ac
    Hydroxytyrosol acetate
• 10-H-Ol agl
    10-H-oleuropein aglycon
• LDL-C
    Low-density lipoprotein
• Lig agl
    Ligstroside aglycon
• LLE
    Liquid–liquid extraction
• Methyl-D-Ol agl
    Methyl-decarboxylated-oleuropein aglycon
• MS
    Mass spectrometer detector
• Ol
    Oleuropein
• Ol-agl
    Oleuropein aglycon
• OMWW
    Olive-mill wastewater
• Pin
    Pinoresinol
• RI
    Ripeness index
• RP-HPLC
    Reversed-phase high-performance liquid chromatography
• SOD
    Superoxide dismutase
• SPE
    Solid-phase extraction
• TC
    Total cholesterol
• TEBARS
    Thiobarbituric-acid-reactive substances
• TG
    Triglycerides
• TOF-MS
    Time-of-flight mass spectrometer
• Ty
    Tyrosol
• UV-DAD
    Ultraviolet diode array detector
• VOO
    Virgin olive oil