PHYTOTHERAPY RESEARCH

Phytother. Res. (2016)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5625

Bioavailability of Bioactive Molecules from Olive

Leaf Extracts and its Functional Value

Daniel Martín-Vertedor,1* María Garrido,2 José Antonio Pariente,2 Javier Espino2 and
Jonathan Delgado-Adámez1
1
Technological Institute of Food and Agriculture (INTAEX), Extremadura Government, Avda. Adolfo Suárez s/n, 06007, Badajoz,
Spain
2
Department of Physiology, Neuroimmunophysiology, and Chrononutrition Research Group, Faculty of Science, University of
Extremadura, Avda. Elvas s/n, 06006, Badajoz, Spain

Olive leaves are an important low-cost source of bioactive compounds. The present study aimed to examine the
effect of in vitro digestibility of an olive leaf aqueous extract so as to prove the availability of its phenolic com-
pounds as well as its antioxidant, antimicrobial, and anticancer activity after a simulated digestion process. The
total phenolic content was significantly higher in the pure lyophilized extract. Phenolic compounds, however,
decreased by 60% and 90% in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively.
Regarding antioxidant activity, it was reduced by 10% and 50% after gastric and intestinal digestion, respec-
tively; despite this fact, high antioxidant capacity was found in both SGF and SIF. Moreover, the olive leaf extract
showed an unusual combined antimicrobial action at low concentration, which suggested their great potential as
nutraceuticals, particularly as a source of phenolic compounds. Finally, olive leaf extracts produced a general
dose-dependent cytotoxic effect against U937 cells. To sum up, these findings suggest that the olive leaf aqueous
extract maintains its beneficial properties after a simulated digestion process, and therefore its regular consump-
tion could be helpful in the management and the prevention of oxidative stress-related chronic disease, bacterial
infection, or even cancer. Copyright © 2016 John Wiley & Sons, Ltd.
Keywords: olive leaf; phenolic compounds; antibacterial activity; antifungal activity; anticancer activity.

toxicity, limited costs, broad availability, and potential

BACKGROUND
health benefits. Extraction is one of the most widely
used operations in food industry. It is mainly utilized
The European Union dominates world production of for obtaining certain desired bioactive components ini-
olive oil (>70%), and it is also the largest consumer. tially retained in a food matrix (Pinelo et al., 2005).
Spain is the European country with the highest produc- Molecules obtained by extraction present important
tion of olive oil (54%), the Spanish olive oils having medical and functional properties (Lee and Lee, 2010;
been recognized as high-quality oils. Extremadura, a Delgado-Adámez et al., 2012a). A wide range of
south-western Spanish region, is the third olive oil pro- by-products derived from food industry have been stud-
ducer in Spain after Andalucía and Castilla-La Mancha, ied for this purpose, the attention being focused on
with about 30 million of olive trees and 53 000 tonnes of those that contain high levels of phenolic compounds.
annual oil production (Fuentes et al., 2015). The by-products obtained from fruit and vegetable pro-
Olive leaves are important for the oil industry cessing (peel, seeds, and stones) are a special source of
because they are low-cost source to obtain bioactive these compounds (Schieber et al., 2001; Delgado-Adámez
compounds and provide an environmental benefit be- et al., 2012b). Similarly, olive cake (also called wet
cause of the exploitation of residues of oil industries pomace) and olive leaf have been considered to be
(Briante et al., 2002). Moreover, olive leaves could pro- an interesting source of phenolic compounds. In this
vide a benefit for society because the intake of products sense, recent studies have investigated the enrich-
rich in polyphenolic compounds leads to the prevention ment of olive oils with a phenolic extract obtained
of chronic diseases, thereby reducing the risk of such from olive by-products, and it was observed that phe-
diseases (El Sedef and Karakaya, 2009). nolic compounds in the enriched oils were signifi-
In recent years, numerous initiatives have opted for cantly increased (Japón-Luján and Luque de Castro,
the study of plant extracts for their application in the 2008).
field of nutrition in order to improve food safety by re- The absorption and bioavailability of phenolics in
placing synthetic additives (Delgado-Adámez et al., humans are controversial. It was widely believed that
2014). Substances derived from foods and plants have polyphenols could not be absorbed intact after oral
recently attracted much attention because of their low ingestion but were hydrolyzed to their aglycones by
bacteria enzymes in the lower gastrointestinal tract. It
was further suggested that the aglycones might then be
* Correspondence to: Daniel Martín-Vertedor, Technological Institute of
Food and Agriculture (INTAEX), Extremadura Government, Avda.
partially absorbed or may undergo further biotransfor-
Adolfo Suárez s/n, 06007 Badajoz, Spain. mation by bacteria. Data on these aspects of phenolics
E-mail: [email protected] are scarce and merely highlight the need for extensive
Received 26 October 2015
Revised 27 February 2016
Copyright © 2016 John Wiley & Sons, Ltd. Accepted 18 March 2016
 D. MARTÍN-VERTEDOR ET AL.

investigations of the handling of phenolics by the gastro- from leaves with ultrapure water (1:10 w/v) for 3 h at
intestinal tract and their subsequent absorption and 60–65 °C, and the extract was filtered and centrifuged
metabolism (Karakaya, 2004). at 21,036 ×g to remove solid particles (Delgado-Adámez
Currently, research in the field has focused the atten- et al., 2014).
tion on understanding the mechanisms of release of cer-
tain food compounds, especially those considered
beneficial for human health. However, it is crucial to Stabilization of aqueous extract. The olive leaf extracts
develop accurate models of digestion that simulate the were stored at
80 °C until they were freeze-dried in a
set of reactions that occur in the interior of the gastroin- lyophilizer (Virtis Company, Mod. Génesis 25 LL,
testinal tract in order to verify the potential bioavailabil- Hücoa-Herlos). The dry extracts obtained were kept away
ity and effectiveness of these products. In vivo methods from the light (at room temperature) in amber-colored
are more accurate and appropriate, but they are slow glass bottles until further analysis.
and often quite expensive. This is why researchers tend
to develop in vitro digestion techniques that pose a
quicker and cheaper alternative, despite the fact that
in vitro methods do not manage to achieve similar levels Simulated gastrointestinal digestion. To study the
of accuracy compared to the in vivo ones because of the behavior of the olive leaf extract during digestion, con-
inherent complexity of the process. Thus, a series of trol (ultrapure water) and olive leaf extract were sub-
in vitro digestion models have been developed to assess jected to an in vitro assay following the method
structural changes, bioavailability, and digestibility of described by Calvo et al. (2012). The in vitro digestion
foods, indicating that in vitro digestion systems are com- was carried out in three phases. First, samples were
mon and useful tools for analyses of foods and drugs exposed to an oral fluid which contained 1 mL of human
(Hur et al., 2011). saliva g
1 of extract. After that, they were mixed at slow
To further support the functional value of olive leaf speed using an Omni Mixer Homogenizer (Ivan Sorvall,
extract and increase the value of the by-products gener- Inc., Norwalk, Conn. USA) during 20 s. A warm bath
ated in the olive industry, the general objective of this was used to maintain the temperature around 37 °C.
research was to assess the antioxidant, antimicrobial, Second, the mix was exposed to simulated gastric fluid
and anticancer effects of olive leaf aqueous extract (SGF) containing pepsin and sodium chloride at low
before and after in vitro gastrointestinal process. pH value. Finally, an intestinal digestion was simulated
by exposing gastric digestion elements to a simulated
intestinal fluid (SIF) containing pancreatin, lipase, as
well as cholic and deoxycholic acids in PBS buffer.
The SGF was prepared according to the United States
MATERIALS AND METHODS Pharmacopeia method (The United States Pharmacopeial
Convention, Inc, 2000) and contained 0.2-g pepsin and
Plant materials. The study was carried out in the Tech- 0.125-g sodium chloride in deionized water to give a final
nological Institute of Food and Agriculture of Extrema- volume of 62.5 mL at pH 1.5.
dura (INTAEX). The olive leaves were picked up from The mix of olive leaf extract (1 g) and saliva was
the experimental olive (Olea europaea L.) orchard added to 3.6 mL of SGF and the final mixture (pH 2.2)
maintained by the Researcher Center ‘Finca La was stirred for 20 min at 37 °C in the digester (K-349;
Orden-Valdesequera’ (Badajoz, Spain) within the limits Buchi, Flawil, Switzerland). After that, pH was adjusted
of the olive-growing area ‘Tierra de Barros’. Samplings to 6.5 with NaOH to inactivate pepsin. The SIF was pre-
of the olive leaves of ‘Arbequina’ cultivar were carried pared in 0.1 M PBS buffer (100 mL, pH 3.4) containing
out in the morning, taking samples randomly, in perfect 20-mg pancreatin, 5-mg lipase, 10 mM cholic acid, and
sanitary conditions. Specifically, 500 g of olive leaves 10 mM deoxycholic acid (Lee et al., 2003). Then,
was taken from different parts of the central area of 3.6 mL of SIF was added to the resulting product of
the olive tree in three times. The samples were collected the gastric digestion, adjusting the pH to a final value
at spotted stages of maturation, using the subjective of 6.5. The mixture was stirred for 20 min at 37 °C to
evaluation of color of the skin and flesh. After harvest- complete the intestinal digestion.
ing, all samples were immediately transported to the After each digestion step (gastric and intestinal), the
INTAEX laboratory, using ventilated storage trays to digestion mixtures were centrifuged at 21,036 ×g for
avoid compositional changes. The olive leaves were 10 min at 4 °C to remove solid particles. Finally, they
vacuum-packed (Gustav Müller VS 100, Germany) in were quickly stored in amber-colored glass bottles at
plastic bags and stored at
80 °C until further use.
80 °C until further analysis.

Extraction of bioactive compounds. Olive leaves used in Chemicals and instruments. HPLC analysis was per-
this study were removed from leaders by hand. Olive formed using an Agilent Technologies series 1100
leaves were partially dried in a conventional oven (Agilent Technologies S.L., Madrid, Spain) system
(model 210, Selecta® P, Spain) for 12 min at 120 °C to equipped with an automatic injector and a diode array
obtain the most suitable material to perform the subse- UV detector. HPLC mobile phase was provided by
quent extraction. Samples were mixed two or three Calbiochem (USA and Canada). Folin-Ciocalteau re-
times while they were in the oven to facilitate drying. agent, caffeic acid, and sodium carbonate were procured
Dried samples were ground in a domestic knife mill from Panreac (Barcelona, Spain). MTT (3-[4,5-dimethyl-
and were sieved to select particles between 0.5 and thiazol-2-yl]-2,5 diphenyl tetrazolium bromide), DMSO,
3.0 mm. Finally, bioactive compounds were extracted and 6-hidroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic
Copyright © 2016 John Wiley & Sons, Ltd. Phytother. Res. (2016)
 POTENTIAL HEALTH BENEFITS OF OLIVE LEAF EXTRACT

acid (Trolox) were acquired from Sigma-Aldrich (CECT) of Valencia University. The bacterial strain
(Madrid, Spain), whereas 2,2′azobis-(3-ethylbenzothiazo- used in the assay of the antimicrobial activity of the
line-6-sulfonic acid) (ABTS) was obtained from Fluka extracts was Escherichia coli 45.
Chemicals (Madrid, Spain). All enzymes for in vitro diges-
tion experiments were purchased from Sigma-Aldrich. Determination of antimicrobial activity. The antimi-
All chemicals for the digestive fluids were obtained from crobial activity was studied following the procedure pre-
Merck (Madrid, Spain). An UV–Vis spectrophotometer viously established by Delgado-Adámez et al. (2012a).
model HP8453 (Agilent, Madrid, Spain) was used for These analyses were carried out in both the olive leaf
antioxidant potential and total polyphenol content analy- dry extract, and the extracts digested in the simulated
ses. An automatic microplate reader (Infinite M200; model (gastric and intestinal). The extracts were first
Tecan Austria GmbH, Groedig, Austria) was utilized solved in water and then diluted to the highest concen-
for the cytotoxicity assay. tration (0.1% v/v) to be tested. Afterwards, serial dilu-
tions were made in a concentration range from 0.1%
to 0.001% (v/v). The target microorganism was cultured
Individual phenolic compound determination. The in Mueller-Hinton broth (MHB) at 37 °C for 24 h. After
individual phenolic compounds was analyzed in both that, the suspensions were diluted with 0.5 McFarland
the olive leaf extract and the extract digested in the sim- standard turbidity and diluted again (1/1000 ratio) in
ulated model (gastric and intestinal) following the MHB. It was added 180 μL of MHB containing diluted
method proposed by González-Gómez et al. (2009) with bacteria (~105 Colony-Forming Units (CFU) mL
1)
some minor modifications. Chromatographic separation and 20 μL of the different solutions of the extracts per
was accomplished with a Phenomenex C18 HPLC well in 96-well microtiter plates. A positive control (con-
column (150 mm × 4.6 mm, 5 μm) heated to 35 °C. The taining inoculums but no extracts) and negative control
mobile phase used for the separation was composed of (containing extracts but no inoculums) were included
aqueous TFA 0.1% (A) and acetonitrile (B) in gradient on each microplate to verify any change in absorbance
mode set as follows: initial conditions, 10% of B; from 0 of the olive leaf extract. The contents of the wells were
to 3 min, 10% of B; from 3 to 15 min, 15% of B; from 15 mixed and the microplates were incubated at 37 °C for
to 20 min the composition was kept constantly at 15% of 24 h under aerophilic conditions. Absorbances at
B; from 20 to 25 min, 18% of B; and from 25 to 40 min, 450 nm were measured at 0 and 24 h in a plate reader.
30% of B. A period of 5 min was necessary for column Turbidity readings were related to bacterial growth.
equilibration. The flow was fixed at 0.500 mL/min for The inhibitory effect was calculated using the following
all the experiments. The injected volume was 5 μL. formula:
Chromatographic data processing was performed
ΔAbsReference - ΔAbsAssay
using Agilent ChemStation software, and the quantifica- %Inhibition ¼ 100
tion was done by external standard method. Phenolic ΔAbsReference
compound quantification was achieved by the absor- where:ΔAbsReference is the increase in absorbance of
bance recorded in the chromatograms relative to exter- control sample.ΔAbsAssay is the increase in absorbance
nal standards, with detection at 280 nm for oleuropein of test sample.
and flavanol, at 320 nm for phenolic acids and
vesbascoside, at 350 nm for flavonoids, and at 360 nm
for flavonols. Luteolin derivatives were quantified as
Anticancer activity assay
milligrams of luteolin 7-O-glucoside, flavanol was quan-
tified in milligrams of epicatechin, and flavonols were
measured in milligrams of quercetin-3-O-rutinoside. Cell culture. Human histiocytic leukemia (U937) cell
The other compounds were quantified as themselves. line (ECACC No. 85011440) was grown in RPMI 1640
medium (Lonza, Barcelona, Spain) supplemented with
2 mM L-glutamine, 10% heat-inactivated fetal bovine
serum, 100 U/mL penicillin and 100 μg/mL streptomy-
Antioxidant activity assay. The capacity of radical scav- cin. Cells were cultured in a humidified atmosphere con-
enging of both the olive leaf dry extract and the extract taining 5% CO2 at 37 °C. Given that previous works has
digested in the simulated model (gastric and intestinal) proven the efficiency of different olive leaf extracts
was assessed by the ABTS• + method (Cano et al., against leukemia cells (Abaza et al., 2007; Fares et al.,
2000). Briefly, 1 mL of the radical cation ABTS was 2011; Samet et al., 2014), we used U937 cells to study
placed in a spectrophotometric cuvette, and 20 μL of whether the olive leave extracts maintained their anti-
the phenolic extract was added. The initial absorbance cancer potential after a simulated digestion.
value at 730 nm was then compared with the
absorbance obtained after 20 min of reaction. The Cytotoxicity assay. The cytotoxic effects of the olive
results were expressed as Trolox antioxidant equivalents leaf extract on leukemic U937 cell line were studied by
(TAE) that were expressed as mmol Trolox kg
1 the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl
extract. The calibration curve was done using different tetrazolium bromide) assay. The cells were seeded at a
Trolox concentrations. density of 5 × 104 cells/well in 24-well plates. The olive
leaf dry extract (1 g) was dissolved in 9 ml distilled
H2O and then serially diluted in a concentration range
Antimicrobial activity assay from 0.1% to 0.001% (v/v). The aqueous extract
digested in the simulated model (both gastric and intes-
Bacterial strains. Bacterial cultures used in this study tinal digestion) was also serially diluted in a concentra-
were obtained from the Spanish type culture collection tion range from 0.1% to 0.001% (v/v). Five microliters
Copyright © 2016 John Wiley & Sons, Ltd. Phytother. Res. (2016)
 D. MARTÍN-VERTEDOR ET AL.

of each concentration of both the aqueous extract and

RESULTS
the digested-like extracts was applied to the wells of a
24-well plate containing sub-confluent cell cultures.
After 24 h of incubation, MTT solution (5 mg/mL) was Phenolic compounds and antioxidant activity in the
then added to each well, and the formazan precipitate olive leaf extract
was dissolved in 200-μL DMSO after 1 h of incubation
at 37 °C. The content of the wells was homogenized on The individual phenolic compounds were quantified
a microplate shaker for 5 min. The optical densities by HPLC using external standard calibration curves
(OD) were measured on a microplate reader at a test for each individual phenolic compound. The linearity
wavelength of 490 nm and a reference wavelength of range of the analytical procedure ranged from 98.5 to
650 nm to cancel out the effect of cell debris. All tests 99.3% of the studied compounds. We used standard
and analyses were run in duplicate, and mean values phenol concentrations and injected the correspondent
were recorded. The cell survival curves were calculated standard solution three times. All calibration curves,
as percentage of control values (untreated samples). which were obtained as a function of the integrated
peak area, were linear throughout the studied range,
with determination coefficients (r2) > 0.99 for all com-
Statistical analysis. For statistical studies, SPSS 17.0 soft- ponents. The results obtained in the evaluation of the
ware was used (SPSS Inc. Chicago, IL, USA). All anal- individual phenolic content are shown in Table 1.
yses were done in quintuple, unless otherwise indicated. Significant quantitative differences were observed in
Data were expressed as means ± SEM and were ana- a wide number of phenolic compounds in the olive
lyzed using a one-way analysis of variance (ANOVA) leaf extract. Thus, it was found that the most
followed by Tukey’s multiple range test to analyze the abundant compound in the olive leaf extract
effect of the digestion process. The effect of the dilution was oleuropein. In fact, oleuropein represented more
was analyzed in the antimicrobial and anticancer assays than 50% of the total identified compounds. Apart
as well. The significance level was set at p < 0.05. from oleuropein, bioactive molecules such as

Table 1. Composition of the individual phenolic compounds and antioxidant activity obtained in the olive leaf aqueous extract and after
gastric and intestinal digestion in vitro

Olive leaves Simulated gastric Simulated intestinal

1
Phenolic compounds (mg/kg ) extract digestion digestion

Phenolic acids
Gallic acid 1.73 ± 0.14 nd nd
Vanillin 0.47 ± 0.15 nd nd
p-Cumaric acid 0.81 ± 0.16 nd nd
Ferulic acid nq nq nq
Chlorogenic acid 30.72 ± 0.82 nd nd
b a
Caffeic acid 201.5 ± 21.2 18.7 ± 0.19 nd
Phenolic alcohol
c b a
Hydroxytyrosol 10 924.7 ± 541.6 3195.1 ± 457.6 1124.4 ± 142.8
c b a
Tyrosol 1681.2 ± 111.4 369.8 ± 71.6 170.2 ± 11.0
Secoiridoid derivatives
c b a
Oleuropein 22 541.8 ± 1057.6 4050.6 ± 122.5 1447.9 ± 155.5
Flavonoids
c b a
Luteolin 7-o-glucoside 3741.9 ± 120.1 698.7 ± 44.8 268.9 ± 17.6
c b a
Apigenin 7-o-glucoside 2147.1 ± 124.9 587.4 ± 11.1 257.4 ± 19.5
c b a
Rutin 357.4 ± 14.7 50.7 ± 11.6 25.7 ± 8.8
Hidrocinamic derivatives
c b a
Verbascoside 914.4 ± 20.7 196.4 ± 17.1 79.6 ± 14.6
Lignans
Pinoresinol nd nd nd
Acetoxypinoresinol nd nd nd
Flavanol
Epicatechin 21.73 ± 0.37 nd nd

Flavonols
Quercetin-3-O-rutinoside 14.89 ± 0.67 nd nd
b a
Quercetin-3-O-galactoside 30.57 ± 1.55 11.7 ± 8.6 nd
Kaempferol 5.58 ± 0.42 nd nd
c b a
Antioxidant activity (mmol Trolox l-1 extract) 15.6 ± 2.01 10.3 ± 0.15 8.4 ± 0.01

Results are expressed as mean ± SEM of three sample replicates. Different small letters in the same row indicate significant statistical differ-
ences (Tukey’s test, p < 0.05) among treatments. nd: not detected; nq: not quantified.

Copyright © 2016 John Wiley & Sons, Ltd. Phytother. Res. (2016)
 POTENTIAL HEALTH BENEFITS OF OLIVE LEAF EXTRACT

hydroxytyrosol, luteolin, apigenin, and verbascoside

were found at high concentration in the extract.
Phenolic acids such as vanillin, p-cumaric acid, and
caffeic acid were minor compounds, provided
its concentration corresponded to 0.5% of total
phenolics.
Nevertheless, as depicted in Table 1, the amount
of individual phenols showed significant variations
(p < 0.05) after the different experimental conditions.
Thus, the effect of digestion was evaluated by quan-
tifying the phenols in the aqueous micellar phases
after the gastric and intestinal digestion steps, which
was calculated back to 1 g of olive leaf extract. These
findings were compared with the values obtained for
the undigested extract in order to address the influ-
ence of the digestion process into the aqueous olive Figure 1. Percentage of inhibition in the aqueous olive leaf extract
leaf extract. Following the digestion process, on before and after gastric and intestinal digestion in vitro in
average the initial amount of phenolic compounds Escherichia coli.. Results are expressed as mean ± SEM of three
was reduced by 60% after being subjected to the sample replicates. Different capital letters over the columns indi-
action of gastric acids in vitro. Therefore, 40% of cate significant statistical differences (Tukey’s test, p < 0.05)
along the dilutions in each treatment. Different small letters over
the phenolic compounds would be available to be the columns indicate significant statistical differences (Tukey’s
absorbed by the organism and would enter into test, p < 0.05) among treatments in each dilution.
the intestinal tract. The lowest values found
corresponded to olive leaf extract subjected to intes-
tinal digestion where there was a reduction of 90%
of phenolic compounds with respect to the olive leaf
fresh extract. That means that 10% of phenolic com-
pounds would be available to be assimilated in the
intestinal tract.
Concerning the antioxidant activity of the olive
leaf extract, it was measured by the ABTS method
and reached a concentration of 15.60 mmol Trolox
kg
1 extract (Table 1). Interestingly, after gastric
and intestinal digestions we found a high antioxidant
capacity, i.e. 10.30 and 8.40 mmol Trolox kg
1 ex-
tract, respectively, which means that it was reduced
by 34% and 46%, respectively, compared to the fresh Figure 2. Dose-dependent cytotoxic effect of the aqueous olive
olive leaf extract. leaf extract. After 24 h of exposure to the aqueous or the in vitro
digested extracts (gastric and intestinal digestion), their
citotoxicity towards U937 cell line was determined by the MTT
assay. Values are presented as means ± SEM of six independent
Antimicrobial activity of the olive leaf extract experiments and expressed as percentage of control values (un-
treated samples). Different capital letters over the columns indi-
The olive leaf aqueous extract and its in vitro diges- cate significant statistical differences (Tukey’s test, p < 0.05)
tions were screened for their antimicrobial activity along the dilutions in each treatment. Different small letters over
against E. coli at different dilutions (Fig. 1). The the columns indicate significant statistical differences (Tukey’s
test, p < 0.05) among treatments in each dilution.
aqueous extract inhibited almost completely the
growth of E. coli when diluted 1:10 and 1:100, while
the inhibition was around 60% for the dilution
1:1000. Certainly, the chemical composition of the aqueous extract. At the highest concentration tested
olive leaf extract conditioned the antimicrobial effects (1:10), the aqueous extract exhibited a substantial,
observed. Additionally, after in vitro gastric digestion statistically significant (p < 0.05) cytotoxic effect, thus
model, an inhibition superior to 85% was observed in reducing the cell viability by 40%. The intermediate
the first two dilutions (1:10, 1:100), while the inhibi- concentration (1:100) was partially efficient in
tion was higher than 60% in 1:1000 dilution. Finally, diminishing cell viability. In fact, the olive leaf extract
the intestinal digestion produced an inhibition provoked a significant (p < 0.05) diminution in U937
of 60%, 15%, and 5% in the three dilutions, cell viability (~20%). At the lowest concentration
respectively. tested (1:1000), the olive leaf aqueous extract
displayed poor cytotoxicity. More importantly, after
the simulated in vitro digestion, either gastric or in-
Cytotoxic activity displayed by the olive leaf extract testinal, the olive leaf extract retained the cytotoxic
potential. As a matter of fact, at the highest concen-
The olive leaf aqueous extract was also used to assay tration tested (1:10), the olive leaf extract diminished
its cytotoxic activity against U937 cells at different U937 cell viability by 20% and 10% after the gastric
dilutions. As shown in Fig. 2, a general dose- and intestinal digestion, respectively. At lower con-
dependent decrease in the survival of U937 cells centrations (1:100 and 1:1000), the olive leaf extract
was observed after treatment with the olive leaf showed negligible cytotoxicity.
Copyright © 2016 John Wiley & Sons, Ltd. Phytother. Res. (2016)
 D. MARTÍN-VERTEDOR ET AL.

activity than virgin olive oil. For instance, Samaniego

DISCUSSION
et al. (2007) referred high antioxidant activity with
values of 1.00 mmol Trolox kg
1 oil in ‘Picual’ extra-
Olive leaves are a cheap raw material that can be used virgin olive oil, whereas Calvo et al. (2012) presented
as an adequate supply for products with high added values of total antioxidant activity of 1.35 mmol Trolox
value (Briante et al., 2002) and a potential source of nat- kg
1 oil in ‘Morisca’ and ‘Picual’ monovarietal oils. In
ural antioxidants because of its high content in phenolic this sense, some researchers evaluated strategies for
compounds. It is important to note that the use of meth- the development of a virgin olive oil enriched with aque-
anol or hexane as extractants has been previously ous extracts obtained from olive leaf and olive cake in
rejected owing to its toxicity (Japón-Luján and Luque order to increase the dose of phenolic compounds
de Castro, 2008). Thus, we have assayed an aqueous ingested in the diet without the drawback of a high
phenolic-rich extract that could be used as a new prod- calorie intake (Delgado-Adámez et al., 2014). This fact
uct for oil industry, given that it is easily obtained and could be especially appealing for the olive industry
shows feasibility of production. Olive leaves were given that it adds further support to the functional value
selected based on previous research whereby we evalu- of virgin olive oil and, at the same time, increases the
ated different aqueous extracts obtained from olive value of olive by-products. Moreover, European Food
leaves and olive cake so as to determinate the optimal Safety Authority (EFSA) has recently accepted the
conditions for phenolic extraction (Delgado-Adámez olive leaf aqueous extract as a safe product and, there-
et al., 2014). In this way, two variables that potentially fore, the olive leaf extract could be used as food addi-
influence the process, i.e. drying time and temperature, tive. In fact, olive leaf extract contain many potentially
were optimized in a multivariate study, using total phe- bioactive compounds that may have antioxidant, antihy-
nolic compounds as independent variable. We found pertensive, antiatherogenic, antiinflammatory, antican-
that the extract with less drying time, i.e. 12 min, and cer, hypoglycemic, and hypocholesterolemic properties
higher temperature, i.e. 120 °C, stood out with the signif- (De Marino et al., 2014; Fakhraei et al., 2014; Olmez
icantly highest total phenolic content. Nonetheless, the et al., 2015; Kishikawa et al., 2015).
individual phenolic compounds have been also analyzed Interestingly, the digested extracts maintained high
herein in order to characterize the olive leaf extract antioxidant capacity, which may be positively corre-
before and after being subjected to the in vitro gastroin- lated to the amounts of hydroxityrosol, tyrosol,
testinal digestion model (gastric and intestinal steps). and, especially, oleuropein found after gastric and in-
Individual bioactive molecules such as oleuropein, testinal digestion because they are known to possess
hydroxytyrosol, luteolin, apigenin, and verbascoside high antioxidant activity (Somova et al., 2003;
were found at high concentration in the aqueous extract, Carrasco-Pancorbo et al., 2005). This is of outstanding
while others like vanillin, p-cumaric acid, and caffeic importance considering that many functional foods are
acid were minor compounds. All these compounds were currently designed to provide a high intake of bioactive
previously reported to occur in olive leaves (Pereira molecules so as to reduce the risk of diseases associated
et al., 2007a). The quantification of the phenolics pres- with aging and oxidative stress (Giugliano and Esposito,
ent in the aqueous extract revealed a high amount of 2005; Covas, 2008). Nevertheless, despite the fact that
these compounds that was considerably superior to the the preparation of olive leaf extract may result in an
values found in hydromethanolic extracts of the same increase in the bioavailability of phenols in plasma, these
and others olive cultivars, as previously reported promising results should be further confirmed in vivo by
(Meirinhos et al., 2005). Besides, lignans such as the determination of the phenolic metabolites (glucuro-
pinoresinol and acetoxypinoresinol were not detected, nide, methylated, and sulfated derivatives) in plasma sam-
which is in accordance with the results found by ples after the consumption of olive leaf extract. Indeed, the
Paiva-Martins and Gordon (2001). Concerning the knowledge of phenolics’ bioavailability is essential to
in vitro digestion processing, most of the phenolic understand their conjugations and bioactivities in the
acids present in the olive leaf aqueous extract, except organism. Thus, there is evidence that when phenolics
for caffeic acid, were found in both the SGF and the are absorbed in the free form, their absorption and conju-
SIF in their free form, which is rather unstable during gation follow the same pathways as that of flavonoids. In
gastric digestion. More importantly, the remaining addition, phenolic metabolites can retain a strong antioxi-
amount of phenolic compounds after gastric and duo- dant activity and might still exert a significant antioxidant
denal digestion was very significant, especially the con- action in vivo (Heleno et al., 2015).
centration of oleuropein, which is the most abundant As for the assay of antimicrobial activity, the entire
phenol in olive leaves (Japón-Luján and Luque de extracts were used because they may be more beneficial
Castro, 2008). This fact could have great interest than isolated constituents because a bioactive individual
because of the protective effects of oleuropein against ox- component can change its properties in the presence of
idative damage (Paiva-Martins and Gordon, 2001). other compounds present in the extracts (Borchers
Indeed, the ingestion of olive leaf extracts with a high con- et al., 2004; Lee and Lee, 2010). Herein, we proved that
tent of phenolics could be desirable for its use with preven- not only the aqueous extract but also the digested
tive and/or therapeutic purposes (Somova et al., 2003). extracts were able to strongly inhibit the growth of E.
Regarding the antioxidant activity of the olive leaf coli. Other researchers have also reported antimicrobial
extract tested herein (roughly 15 g Trolox kg
1 extract), capacity of leaf phenolics, such as walnut leaves (Pereira
it is clearly higher compared to those previously found et al., 2007b), olive leaves (Pereira et al., 2007a; Lee and
in plum leaf extracts, which ranged from 5.08 ± 0.44 to Lee, 2010), plum leaves (Delgado-Adámez et al.,
1.85 ± 0.44 g Trolox kg
1 extract (Delgado-Adámez 2012b), and hazelnut leaves (Oliveira et al., 2008). The
et al., 2012b). Besides, when compared to virgin olive high content of oleuropein and other phenolic com-
oil, our olive leaf extract had 15-fold higher antioxidant pounds identified in the extract, which contributed to
Copyright © 2016 John Wiley & Sons, Ltd. Phytother. Res. (2016)
 POTENTIAL HEALTH BENEFITS OF OLIVE LEAF EXTRACT

its antioxidant activity, might contribute to its antimicro- and/or cell killing abilities of different olive leaf extracts
bial properties as well (Sudjana et al., 2009; Lee and against leukemia cells (Abaza et al., 2007; Fares et al.,
Lee, 2010). In particular, oleuropein, has exhibited anti- 2011; Samet et al., 2014), none of them has proved
bacterial activity against a wide variety of Gram-positive whether the extracts maintained their anticancer prop-
and Gram-negative human pathogenetic bacterial erties once they entered the gastrointestinal tract.
strains (Pereira et al., 2007a; Sudjana et al., 2009), anti- Therefore, olive leaf extracts warrant further investiga-
fungal properties (Aziz et al., 1998), as well as antiviral tion into their potential antileukemic benefits.
actions mostly against enveloped viruses (Ma et al., Overall, our results suggested that the olive leaf aque-
2001; Micol et al., 2005). Similarly, chlorogenic acid has ous extract, which possessed high content in phenolic
been shown to have strong antimicrobial activity compounds, could be absorbed by the gastrointestinal
(Davidson and Branen, 1981), and is usually present in tract, and its regular consumption may be helpful in
olive leaf and drupe extracts (Brahmi et al., 2013). The the management and the prevention of oxidative
active portion of chlorogenic acid, according to stress-related chronic diseases, bacterial infection, or
Grodzinska-Zachwieja and Kahl (1966), is caffeic acid, cancer. In fact, even after simulated digestion, the olive
a hydroxycinnamic acid. Several hydroxycinnamic acid leaf extract depicted in vitro antioxidant, antimicrobial,
derivatives have been found to have antimicrobial and antitumor activities.
effects against several microorganisms, including E. coli
(Lee and Lee, 2010). During the digestion these com-
pounds are reduced, which may be directly correlated
with the lower antimicrobial activity found with respect Acknowledgements
to that obtained in aqueous extracts. Anyway, our find- This work has been financed by European Regional Development
ings demonstrated that the use of olive leaves as Funds (ERDF). This study has been carried out with financial sup-
nutraceuticals may diminish the risk of E. coli infections, port from Government of Extremadura (project 6/12). J. Espino
particularly in the gastric and intestinal tract as observed and M. Garrido hold a research post-doctoral fellowship from
with 1:10 and 1:100 dilutions, which is probably because Government of Extremadura (jointly financed by the European
of the protective actions provided by its phenolic Regional Development Fund (ERDF); ref. PO14011 and PO14013,
compounds. respectively). The authors wish to thank to the Research Centre
In relation to the anticancer assay, our findings sug- ‘Finca la Orden-Valdesequera’ by the products of the field experi-
ment cultivars.
gested that the digested olive leaf extract could preserve
its anticancer potential against human histiocytic
leukemia U937 cells, as observed after the in vitro diges-
tion processing, thereby indicating that the extract may Conflict of Interest
be able to act at systemic level. Despite the fact that pre-
vious research has already tested the anti-proliferative The authors have no conflict of interest to declare.

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