沙漠研究 22-1, 61 -64 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－Valorization of Olive Processing By-Products: Characterization, Investigationof Chemico-Biological Activities and Identification of Active Compounds
Zouhaier BOUALLAGUI1), Mohamed BOUAZIZ1), Junkyu HAN2), Maher BOUKHRIS1), Ghayth RIGANE1),
Ines FRIHA1), Hedya JEMAI1), Ines FKI1), Héla GHORBEL1), Hiroko ISODA2) and Sami SAYADI*1)
Abstract: The valorization of food processing by-products, wastes and effluents is a challenging opportunity for the sustainable andcompetitive development of several relevant industrial sectors. One of the best examples of a biorefinery applied to plant processing
wastes is that of the olive oil sector by-products including leaves and olive mill wastewaters (OMW). Management of OMW, in
particular, has received much attention over recent years including all possible strategies, i.e. treatment, disposal, recovery and reuse.
Besides being a serious environmental problem, OMW can also represent a precious resource of potentially valuable molecules. This
later approach appears to be the new frontier in olive processing by-products valorization.
The present paper will review the scientific works done in our laboratory concerning the valorization of olive processing
by-products mainly OMW and olive leaves through the recovery of valuable molecules. Analytical chemical techniques such as
solvent extractions and characterization of extracts in respect with their phenolic content were performed. Different methods werealso used to investigate the activities of different extracts and fractions. Therefore, the antioxidant assays using DPPH
(1,1’-diphenyl-2-picrylhydrazyl), beta-carotene, ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)) and bioassays
including anti-tumoral activity, and in vivo activities in animal models were carried out. Besides, analytical chromatography andspectroscopic techniques were used by our group either for a deep characterization of active fractions or for the identification of newmolecules.
Key Words: Antioxidants, Bioactivities, Olive by-products, Polyphenols, Valorization

1. Introduction
Olea europaea L. is a typical tree widely cultivated for oil
production in the Mediterranean area.
Tunisian olive
plantation counts about 60 million trees covering more than
1.600.000 ha. The sector of olive oil accounts for around 47%
of agro-alimentary exports and 5.5% of total exports of thecountry. Olive leaves and olive mill wastewater, resulting
from the water use during oil extraction, as the two main olive
processing by-products, have attracted considerable attention as
valuable resources of bioactive molecules.
OMW and olive leaves are considered as an inexpensive
source of highly valuable molecules. These molecules mainly
belong to the family of polyphenols. The most studied olive
polyphenols are oleuropein, a secoiridoid group member, and its
hydrolysis derivative; hydroxytyrosol (2 (3,4-dihydroxyphenyl)
ethanol) (Obied et al.2005).
In respect with this topic, our research team has been
implicated, during the last few years, into a local and a
Mediterranean strategy in order to establish practical methods
devoted to a rational management of olive processing wastes inparticular OMW and olive leaves. Here we are reviewing themajority of our work in relation with the development of asustainable valorization process of the aforementioned
by-products.

2. Olive Mill Wastewater as a Source of Valuable
Molecules
It is well admitted that OMW represents a great challenge as
an environment-harming factor in olive oil producing countries.
Chemically, OMW is characterized by a high organic load thus
limiting its biological treatment. Phenolic compounds werefound as major contributors to the toxicity and the antibacterial
activity of OMW (Sayadi et al., 2000). However, these
phenolic compounds are endowed with several biologicalactivities such as antioxidant properties (Visioli et al., 1999).
OMW has attracted a considerable attention as a source ofnatural polyphenols. Indeed 98% of olive fruit biophenols arelost in OMW during oil extraction (Rodis et al., 2002). Table
1 illustrates the major phenolic monomers retrieved in OMWwith their spectroscopic data determined using gas
chromatography coupled with mass spectrometry.

* Corresponding Author: sami.sayadi@cbs.rnrt.tn
Route de Sidi Mansour Km 6, BP "1177", 3038, Sfax – Tunisia1) Laboratoire des Bioprocédés Environnementaux, Pôle d’Excellence Regional AUF, (PER-LBP),Centre de Biotechnologie de Sfax.
2) Alliance for Research on North Africa (ARENA), Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.

Table 1. Mass spectrometry data of the Major Phenolic Monomers.
Identified in the crude OMW.
Compounds

mass spectrometry

Concentration in

Data (m/z )OMW (mg/l)

Hydroxytyrosol

370; 267; 193; 179.

1433.4

Tyrosol

282; 267; 193; 179;

851

Oleuropein

163; 149; 103.
3,4-dihydroxyphenyl384; 369; 311; 296;

acetic acid281; 252; 179; 164;

87.9

147
p-hydroxyphenyl

296; 281; 252; 179;

acetic acid
Caffeic acid

164; 147; 133.
396; 381; 307; 293;

274

Fig. 1. HPLC chromatogram of an olive leaf extract. Detection wasperformed at 280 nm.

321249; 233; 219; 191.
p-coumaric acid308; 293; 249; 219;

298

203; 179; 175; 139;
115
Ferulic acid338; 297; 282; 267

95253; 223; 207; 193;
179; 165; 141; 126;

Fig.2. Oleuropein chemical structure.

89

Owing to the increased preference for natural foodingredients, which are generally believed to be safer and lesssubject to hazards compared to synthetic additives, our researchteam has developed a convenient method aiming the recoveryof valuable molecules from OMW as natural agro-industrial
waste.
Particular emphasis was made to recoverhydroxytyrosol.
To achieve this goal, a continuous
counter-current liquid-liquid extraction unit was set. This unit
allowed extracting more than 85% of the hydroxytyrosol
originally present in the OMW (Allouche et al., 2004).

3. Olive Leaves a Raw Material for the Recovery of
Bioactive compounds
The work done in relation with the valorization of olive
leaves focused at first on the identification and characterisationof major polyphenols in particular oleuropein, flavonoids andhydroxytyrosol. The most important fact is that olive leaves
were found to contain higher amounts of oleuropein reachingnearly 6.5 g per 100 g fresh olive leaves (Bouaziz and Sayadi,2005). Figure 1 shows an HPLC UV-spectrum of olive
leaves extract where oleuropein is the major phenolic
compound.
In addition to OMW, olive leaves could also be a natural
source of hydroxytyrosol due to their richness in oleuropein.
Indeed, oleuropein is an ester of hydroxytyrosol and theglucosylated form of the elenolic acid (Fig. 2). The hydrolysisof an ester molecule yields a molecule of hydroxytyrosol.
Hence, olive leaves represent a raw material for the preparationof either hydroxytyrosol rich extract or even highly purified
fractions. Both chemical (acid) and enzymatic hydrolysis wasapplied to oleuropein rich extracts. So far, hydroxytyrosol was
the major compound of the hydrolyzates (Bouaziz and Sayadi,2005; Bouaziz et al., 2006).

4. Use of Olive Leaves and OMW Compounds for theStabilisation of Edible Oils
Olive and husk refined oils are gaining increasing
importance in food industry. However, these oils are highly
susceptible to oxidation. The loss of nutritional quality and theproduction of undesirable off-flavours are the principal markers
of lipid peroxidation in oils. To overcome this problem,compounds recovered from OMW were used as substituents tosynthetic food additives (BHT and BHA).
Through the evaluation of the antiradical and antioxidant
activities of the OMW extract and pure compounds identified inthis extract, our team showed that hydroxytyrosol and3,4-dihydroxyphenyl acetic acid had the highest radicalscavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical andthe highest antioxidant activities using the b-carotene linoleate
assay (Table 2) (Fki et al., 2005). When incorporated in oil
matrix, OMW extract and pure phenolic compounds resulted in
an interesting protecting effect of refined olive and husk oils
against induced lipid peroxidation.
Similarly olive leaves extract (oleuropein rich extract) andolive leaves hydrolyzate extract (hydroxytyrosol rich extract)
were studied in oil matrix using olive and husk refined oils
(Bouaziz et al., 2008). Authors concluded to a greatTable 2. Characterization of OMW polyphenols and evaluation of
their antioxidant activities.
OM W extract

IC50 (µg/ml)
1.2

bleaching (h)>236Hydroxytyrosol
Tyrosol

0.57
10.9>236
122

3,4-Dihydroxyphenyl acetic acid
p-Hydroxyphenyl acetic acid0.64
12.2>236
188

Caffeic acidp-Coumaric acid

0.87
9.5>236
148

Ferulic acid
BHA

1.21
0.91>236>236

BHT

0.89>236

Table 3. Antidiabetic and antioxidant activities of polyphenols
recovered from olive leaves. PG (plasma glucose); HG(hepatic glycogen); TC (total cholesterol); TBARS(ThiobarbituricAcid-Reactive Substances).
Treatment

PG(µg/dl)HG(mg/g)TC(mg/dl)

TBARS(M mol/100mg)

Control

1.05

10.6

98

12

Diabetic control
Oleuropein
Hydroxytyrosol

2.9
1.5
1.55

616
14.6

168
105
107

17.2
13.1
12.8

Table 4. Effect of olive leaves extract treatment on cell cycle
distribution in human breast cancer cells MCF-7; OLE:
olive leaves extract.
Cell cycle distribution

stabilizing effect of refined oils with both olive leaves andhydrolyzate extracts.

5. In vivo Bioactivities of Olive Compounds
5.1. Anticholesterolemic effects of olive compounds in rat
model
Atherosclerosis is the principal cause of pathogenesis of the
myocardial and cerebral infractions thus being the leading
cause of death worldwide. Hypercholesterolemia resulting
from modified lipid metabolism is being the inducing factor ofatherosclerosis. Therefore it would be of interest to findanti-hypercholesterolemic molecules or preparations. Our
team has investigated the possible implication of olive
compounds in the reduction of hypercholesterolemia in ratmodels. OMW extract and hydroxytyrosol were tested (Fki etal., 2007; Jemai et al., 2008). These authors have showed thatphenolic rich extract from OMW and hydroxytyrosol
recovered from olive leaves or OMW had very pronounced
hypocholesterolemic effects. Indeed these treatments wereable to reduce both total circulating cholesterol, low density
lipoprotein-cholesterol (LDL-C) and to increase the level ofhigh density lipoprotein-cholesterol (HDL-C) in serum. In
addition olive polyphenols favorably modified the antioxidant
system within liver, heart and kidneys. Such later activities
were suggested to be the origin of the hypocholesterolemic
activity of olive phenolic compounds.
5.2. Hydroxytyrosol and Oleuropein could haveanti-diabetic activities
Diabetes has been shown to implicate diverse andcomplicated mechanisms correlated with an overproduction offree radicals and many other disorders exerting deleterious
effects on organs acting in the glycemia regulation. Due totheir antioxidant activities, olive polyphenols were suggested tohave anti-diabetic potential. For this reason hydroxytyrosol
and oleuropein were recovered from olive olives and wereG0/GA

SG2/M

39.9
46.9
53
53.1

33.3
27.6
24
23.1

26.1
24.9
22.4
23.2

Treatment
Control
OLE 2000µg/ml
OLE 2400µg/ml
OLE 2800µg/ml

investigated in vivo in rat model for their potential to prevent
alloxan induced diabetes (Jemai et al., 2009). Authors havefocused on the analysis of the glucidic and oxidative stressparameters (Table 3). Olive leaves compounds significantly
reestablished blood circulating glucose and hepatic glycogen inanimals treated with alloxan. This phenomenon was coupledwith the restoration of the total cholesterol in serum and thethiobarbituric acid reducing substances in the liver.

6. Bioactivities of Olive Leaves Extract in Cancer Cells
Model
Recently we have investigated the activities ofhydroxytyrosol rich extract from olive leaves against theproliferation of cultured cancer cells. Breast cancer cells
MCF-7 were used as cancer model. Anti-proliferation assays
using MTT and neutral were performed (Bouallagui et al.
2011).
The studied extract showed a cell growth inhibiting effectthrough the arrest of the division cell cycle at G0/G1 phase(Table 4). Such growth arrest was the consequence of thereduction of the cyclinD1 level. At the up-stream level,
cyclinD1 protein level decrease was the result of decreasinglevel of the peptidyl prolyl cis-trans isomerase Pin1 which is
implicated in many physiological processes.

7. Conclusion
The bioactivities exerted by olive polyphenols highlighted
once again the olive tree by-products, including both olive
leaves and OMW, as a natural source of antioxidants whichcould have advantageous applications to prevent oxidative stressrelated disorders. Thus, processes aiming the recovery ofantioxidant polyphenols from olive by-products will be a
promising alternative for the valorization of such agro-industrialwastes.

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