10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Research Article
Received: 11 December 2018 Revised: 14 March 2019 Accepted article published: 29 April 2019 Published online in Wiley Online Library: 3 June 2019

(wileyonlinelibrary.com) DOI 10.1002/jctb.6049

Statistical optimization and kinetic analysis

of the extraction of phenolic compounds
from olive leaves
George K Lamprou, Anestis Vlysidis,* Konstantinos Tzathas
and Apostolos G Vlyssides

Abstract
BACKGROUND: Olive leaves are considered to be one of the most abundant agricultural waste source rich in phenolic
compounds. A new method for extracting the phenolic compounds contained in olive leaves was developed in this study. The
method was based on the addition of sulfuric acid (H2 SO4 ), hydrolyzing the olive leaves and producing extracts rich in phenolic
compounds. Experimental runs were carried out according to a central composite design (CCD) comprising the most important
factors that affect the extraction process.

RESULTS: The factors examined were extraction temperature, the addition of H2 SO4 and the dilution of olive leaves with water.
Maximum total phenolic content (TPC) was 86.4 mg GAE g –1 dry weight basis (db) at 31.9 ∘ C, 6.4% H2 SO4 (v/w) and 25.2 L : S
(v/w). Validation runs on the optimum were very close to model predictions for TPC. Optimum oleuropein was 43.1 mg g−1 db
at 40 ∘ C, 2.0% H2 SO4 (v/w) and 30 liquid : solid (v/w). Finally, the kinetics of TPC and oleuropein were studied at the optimum
conditions designated by the CCD.

CONCLUSION: In this study, a new cost-effective extraction process of phenolic compounds from olive leaves was examined
as the developed process employs low-value chemicals (i.e. H2 SO4 ) in small amounts offering through its simplicity the right
conditions to scale this process up in industrial applications integrated within olive mill facilities.
© 2019 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: statistical optimization; phenolics; kinetics; olive leaves; oleuropein

INTRODUCTION
Phenolic compounds have attracted the attention of the research
community due to their benefits for human health. Phenolic
compounds are secondary metabolites whose physical role is
to provide protection in plants from pathogens, predators and/or
stress caused by abiotic factors such as hydric deficiency, salinity,
poor soil fertility and climatic conditions.1 The color and sensory
characteristics of a plant’s tissues or fruits also are correlated with
their phenolic profile.2
Research into applications for phenolic compounds is vig-
orous and has attracted various industrial sectors. In 2012, EU
established a list of permitted health claims made about foods,
based on scientific research by EFSA, the European Food Safety
Authority.3 Phenolic compounds already are used in medicine,
and the cosmetics and pharmaceutical industries. Recent research
augments their applications in more potential uses such as
the production of food packaging materials4 and as biological
fungicides.5 Moreover, due to recent changes in EU legisla-
tion, research on their use as food additives is expected to
become more systematic. In 2017, the EU published regulation
the market as a novel food ingredient intended for the general
population.6
Phenolic compounds may be detected in all tissues of a plant,
but are especially abundant in olive fruits, olive oil, olive mill
wastes (either solid or liquid) and olive leaves.7 The latter is con-
sidered one of the most abundant agricultural waste sources that
is rich in phenolic compounds.7 The potential supply of olive leaves
is high and in continuous expansion, because they may be col-
lected at two different points in the growth cycle: as remains
from pruning olive trees and/or as by-product generated from
the olive oil extraction process. The amount of olive leaves col-
lected from pruning is estimated to be 25 kg per tree.8,9 However,
in field conditions the quantity of leaves from pruning cannot be
determined precisely because it depends on many factors, the
most important of which are the selected pruning method, the
∗ Correspondence to: A Vlysidis, Laboratory of Organic Chemical Technol-
ogy, School of Chemical Engineering, National Technical University of Athens,
Zografou, Athens 157 80, Greece. E-mail: anestisvlysidis@gmail.com
School of Chemical Engineering, National Technical University of Athens, Iroon
457

2017/2373, which authorizes the placement of hydroxytyrosol in Polytechneiou 9, Athens, Greece

J Chem Technol Biotechnol 2020; 95: 457–465 www.soci.org © 2019 Society of Chemical Industry

www.soci.org
age of tree, the cultivated variety and the climatic conditions. In
olive mill plants, the generated amounts of olive leaves are ≈4% of
the processed olive fruit.10 According to the global production of
olive oil from 2011 to 2014, 15.5 Mt of olive fruits are processed
every year (FAO, 2014) leading to the generation of 0.62 Mt of
olive leaves. Unlike olive mill wastes, which are very toxic efflu-
ents and difficult to undergo any conventional treatment, olive
leaves consist of a clean solid industrial side stream that can be
handled easily and used for extracting high-added-value prod-
ucts such as phenolic compounds.11 Currently, olive leaves are
usually spread on the land by local farmers in order to enrich
the organic matter of the soil. Alternatively, they are used as
animal feed.12
The phenolic content of an olive tree depends on many factors
such as genotype, tissue type, development stage, environmental
conditions and cultural practices.13 Trees grown under the princi-
ples of organic farming had an increased polyphenol content com-
pared to those of conventional farming.14 Total phenolic content
(TPC) varies considerably by tissue and variety. Furthermore, har-
vesting period is a critical factor.13 Other factors, such as the pres-
ence of pests, such as Dacus oleae or weeds may affect the olive
tree phenolic profile.
Solvent extraction is the main method used by most researchers
to retrieve phenolic compounds from olive leaves. According
to the literature, several factors may affect the performance of
the extraction of the phenolic fraction from olive leaves such as
solvent type used, pH, temperature, number of extraction steps,
solvent : solid ratio, and particle size of the solid matrix. Ethanol,
methanol, ethyl acetate, water, hexane, diethyl ether and acetone
have been used as solvents. Methanol seems to be a very effective
solvent, yet its use may result in undesirable toxic residues in the
final extracts.9
The main phenolic compounds contained in olive leaves are
oleuropein and hydroxytyrosol. Due to their numerous health ben-
efits, interest in recovering phenolic compounds from olive leaves
has been increased greatly in the scientific community over the
last decade. Oleuropein is the major phenolic compound in olive
leaves, and large quantities of hydroxytyrosol can be obtained by
hydrolyzing oleuropein.15 Production of hydroxytyrosol requires
enzymatic or chemical hydrolysis of the secoiridoids which contain
it. Enzymatic hydrolysis of oleuropein, occurs via natural mecha-
nisms in the olive tree.16 However, acid hydrolysis is the dominant
mechanism that is employed in the laboratory and industrial pro-
cesses. Derivatives of acid hydrolysis of oleuropein are hydroxyty-
rosol, elenolic acid and glucose.17,18
In this study, we have examined a new extraction method
of phenolic compounds from olive leaves. Our main goal was
to develop a feasible and cost-effective extraction process
that can be implemented even in small- and middle-sized
olive mills. The most important operational conditions of this
extraction process were subject to statistical optimization by
implementing a central composite design where total phenolic
content (TPC) and oleuropein were optimized. Moreover, the
operational conditions that favor the production of hydroxy-
tyrosol were identified. Finally, kinetic studies on the optimum
conditions for the two main outputs (i.e. TPC and oleuropein) were
carried out.
MATERIALS AND METHODS
458
wileyonlinelibrary.com/jctb © 2019 Society of Chemical Industry
10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GK Lamprou et al.
Collection and storage of dried leaves
Fresh green olive leaves were collected from olive trees (Koroneiki
variety) grown in the campus of National Technical University
of Athens (Greece). Olive leaves were washed with deionized water
and air-dried at 20 ∘ C for 3 days. Finally, the olive leaves were stored
in light protected plastic containers for further use.
Extraction procedure
Initially, 10 g of dried olive leaves were added to distilled water. The
leaves were milled using a home blender (Kenwood ch550 multi).
The hydrolysis process was initiated by adding sulfuric acid (H2 SO4 )
98% in the olive leaves/water blend. The composed mixture was
then agitated for 5 h in controlled temperature conditions. At the
end of the 5 h period, the mixture was centrifuged at 6000 rpm
for 10 min. The liquid fraction was further filtered with 0.22-mm
syringe filters and finally stored in the fridge at 4 ∘ C for further use.
In the above-mentioned description, different amounts of water
and H2 SO4 were added and different temperatures were tested
indicated by the CCD (see section Central composite design). The
schematic diagram of the extraction procedure is shown in Fig. 1.
Central composite design
A 33 central composite design was selected to evaluate three of the
most important parameters that affect the extraction of phenolic
compounds present in olive leaves. The selected parameters were
the extraction temperature (X 1 ), the amount of the H2 SO4 (X 2 )
expressed as % volume of acid per weight of dry leaves and the
liquid (L) : solid (S) ratio (X 3 ) expressed as the volume of distilled
water that was used as solvent to the weight of dry olive leaves.
Table 1 shows the ranges of the three factors used in this study.
Selection of the values and ranges of the three optimization
parameters was based on results from preliminary experiments
(results not shown) and on data extracted from the literature
from similar studies that have evaluated the extraction of phenolic
compounds from olive leaves.
Equation (1) was used in order to code the three examined
variables.
Xi − XiCP
xi = (1)
ΔXi
where i is the independent variable (X 1 , X 2 or X 3 ), x i is the coded
value, X i is the real value, X i CP is the real value at the central point
and ΔX i is the step-change value.
The system was predicted by using a second-order polynomial
equation (2):
Yj = 𝛽0 + 𝛽1 x1 + 𝛽2 x2 + 𝛽3 x3 + 𝛽1 𝛽2 x1 x2 + 𝛽1 𝛽3 x1 x3 + 𝛽2 𝛽3 x2 x3
+ 𝛽1 𝛽2 𝛽3 x1 x2 x3 + 𝛽1 𝛽1 x12 + 𝛽2 𝛽2 x22 + 𝛽3 𝛽3 x32 (2)
where Y j are the simulated responses (in our case the simulated
responses are the TPC, hydroxytyrosol, oleuropein and tyrosol), 𝛽 0
is the intercept element, 𝛽 1 , 𝛽 2 and 𝛽 3 are the linear effects of X 1 , X 2
and X 3 , respectively, 𝛽 1 𝛽 2 , 𝛽 1 𝛽 3 , 𝛽 2 𝛽 3 and 𝛽 1 𝛽 2 𝛽 3 are the interaction
parameters of X 1 X 2 , X 1 X 3 , X 2 X 3 and X 1 X 2 X 3 , respectively and
𝛽 1 𝛽 1 , 𝛽 2 𝛽 2 , 𝛽 3 𝛽 3 are the quadratic parameters of X 1 , X 2 and X 3 ,
respectively.
The 11 optimization parameters were estimated by linear regres-
sion minimizing the error between the experimental values with
the simulated responses.
J Chem Technol Biotechnol 2020; 95: 457–465

Extraction of phenolic compounds from olive leaves
Figure 1. Schematic diagram of the extraction methodology followed in this study.
Table 1. The examined levels and ranges of the selected parameters
for the developed CCD
Temperature (∘ C) H2 SO4 % (v/w) L : S (w/v)
Level X1 X2 X3
−1.682 20.0 2.0% 10.0
−1 24.1 3.6% 14.1
0 30.0 6.0% 20.0
1 36.0 8.4% 26.0
1.682 40.0 10.0% 30.0
Analytical procedures
Total phenolic content was determined with the Folin–Ciocalteu
colorimetric method according to a procedure described, where
0.05 mL of sample was initially dissolved in 0.45 mL of distilled
water and then 2.5 mL of 0.2 N Folin–Ciocalteu reagent were
added.19 The mixture was stirred for 3 min and then 2 mL of
saturated sodium carbonate solution were also added. The mixture
was stirred at 30 ∘ C for 1.5 h. The absorbance readings were taken
at 765 nm using a UV-visible spectrophotometer Hitachi U2000
(Hitachi, Tokio, Japan). Gallic acid was used as a reference standard,
and the results were expressed as milligram gallic acid equivalent
(mg GAE) g –1 dry olive leaves (dry weight basis, db).19
The antioxidant activity of the aqueous extracts was measured
according to the DPPH• (2,2-diphenyl-1-picrylhydrazyl) avenging
radical method. Samples of 0.1 mL were diluted in 3.9 mL of DPPH•
solution (31.6 μg mL−1 ). Afterwards, the mixture was shaken and
then stored at room temperature for 90 min, in dark conditions.
As control, a solution of 0.1 mL aqueous methanol (70:30 v/v) in
3.9 mL DPPH• was used. The decrease in absorbance was mea-
sured at 517 nm using a double-beam UV–visible spectropho-
tometer (Hitachi U-2000) 19 .
High-performance liquid chromatography (HPLC) analysis
was performed on an Agilent 1290 Infinity II LC system with a
diode-array detector (DAD) (Agilent Technologies, Santa Clara, CA,
USA) and an Agilent C18 Proshell 120 column (4 μm, 4.6 × 100 mm)
at a flow rate of 1 mL min−1 . The mobile phase consisted of 0.2%
acetic acid in water (A) and acetonitrile (B). The following elution
gradient was used: 2% B at 0 min up to 30% B at 40 to 45 min and
back to 2% B at 45 to 50 min. Injection volume was 20 μL. DAD
signals were recorded at a range of 210 to 360 nm. Calibration
curves were built for all core phenolic compounds expected to be
J Chem Technol Biotechnol 2020; 95: 457–465 © 2019 Society of Chemical Industry
10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org
found in olive leaves extracts such as tyrosol, hydroxytyrosol and
oleuropein.
All analytical standards and chemicals were purchased from
Sigma-Aldrich, whereas all solvents used for chromatographic
analyses were HPLC grade. All samples were analyzed in triplicates.
RESULTS
Experimental results and model predictions
Experimental results of the TPC (expressed as mg GAE g –1 db)
and HPLC analysis including oleuropein, hydroxytyrosol and
tyrosol from the CCD runs are presented in Table 2. In terms
of TPC, the experimental results ranged from 45.8 mg GAE g –1
db to 84.9 mg GAE g –1 db. The latter was found at the coded
values X 1 = 0, X 2 = −1.6818 and X 3 = 0. Also, high values were
obtained at zero level where all runs had a TPC >82 mg GAE g –1
db. Oleuropein had the highest content of the three specific
phenolic compounds extracted. Higher extraction values of oleu-
ropein (>35 mg g−1 db) occurred when lower concentrations of
H2 SO4 were added, but the opposite was true for hydroxytyrosol,
for which higher extraction values (>6 mg g−1 db) took place
when increased amounts of H2 SO4 were added. This behavior is
expected as H2 SO4 breaks oleuropein to hydroxytyrosol, elenolic
acid and glucose. The highest experimental value of oleuropein of
43.2 mg g−1 db occurred at the coded values X 1 = −1, X 2 = −1 and
X 3 = 1, whereas the maximum experimental value of hydroxyty-
rosol (8.3 mg g−1 db) occurred at the coded values X 1 = 1, X 2 = 1
and X 3 = −1.
After the regression analysis and the estimation of the paramet-
ric values, Eqn (2) can be converted to Eqns (3)–(5) for TPC, oleu-
ropein and hydroxytyrosol, respectively:
YTPC = 83.304 + 0.923x1 –0.085x2 + 6.048x3 + 2.32x1 x2
+ 4.493x1 x3 + 2.195x2 x3 –5.57x1 x2 x3 –6.938x12
–2.973x22 –4.350x32 (3)
YOleuropein = 27.653–1.521x1 – − 7.906x2 + 6.317x3
+ 0.311x1 x2 + 1.514x1 x3 + 0.501x2 x3
–1.966x1 x2 x3 + 0.799x12 –0.738x22 –1.252x32 (4)
459
wileyonlinelibrary.com/jctb
 10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org GK Lamprou et al.

Table 2. Experimental results and model predictions of the extraction of phenolic compounds from olive leaves in terms of TPC, oleuropein,
hydroxytyrosol and tyrosol in terms of mg per g dry olive leaves

Model Model Model

predictions predictions predictions
Temperature H2 SO4 % TPC (mg GAE g –1 Oleuropein Hydroxytyrosol Tyrosola TPC (mg oleuropein hydroxytyrosol
Runs (∘ C) (v/w) L:S db) (mg g –1 db) (mg g –1 db) (mg g –1 db) GAE g –1 db) (mg g –1 db) (mg g –1 db)

1 1 1 1 80.2 22.9 7.8 1.6 79.36 23.71 5.85

2 1 1 −1 63.5 9.8 8.3 1.5 65.03 10.98 7.73
3 1 −1 1 76.0 42.1 3.9 2.3 81.64 41.83 2.00
4 1 −1 −1 45.8 23.2 2.3 1.2 53.81 23.24 1.80
5 −1 1 1 80.0 30.6 4.8 2.3 75.03 27.03 3.46
6 −1 1 −1 59.0 15.7 6.1 1.5 56.39 12.49 6.10
7 −1 −1 1 62.8 43.2 2.1 2.2 64.31 38.53 0.76
8 −1 −1 −1 72.9 38.1 2.6 0.8 76.73 33.86 2.66
9 1.6818 0 0 72.3 30.0 0.7 1.1 65.23 27.35 2.77
10 −1.6818 0 0 59.4 24.8 0.3 1.1 62.12 32.46 0.92
11 0 1.6818 0 69.2 11.0 6.5 1.1 74.75 12.26 7.93
12 0 −1.6818 0 84.9 35.1 0 1.3 75.03 38.86 1.24
13 0 0 1.6818 80.5 31.8 0 1.9 81.17 34.73 2.98
14 0 0 −1.6818 65.8 11.5 5.9 0.6 60.82 13.48 5.60
15 0 0 0 82.6 24.6 0 1.4 83.30 27.65 0
16 0 0 0 83.4 25.5 0 1.4 83.30 27.65 0
17 0 0 0 83.4 30.9 0 1.4 83.30 27.65 0
18. 0 0 0 84.2 30.1 0 1.1 83.30 27.65 0
19 0 0 0 82.3 28.0 0 1.2 83.30 27.65 0
a Model predictions of tyrosol are omitted as the predictive capability of this model assumed to be inadequate (R2 = 0.77).

quadratic parameter of H2 SO4 (𝛽 2 𝛽 2 ) also was an important param-

YHydroxytyrosol = 0 + 0.551x1 + 1.988x2 –0.777x3 + 0.455x1 x2
+ 0.358x1 x3 –0.353x2 x3 –0.168x1 x2 x3 + 0.688x12
+ 1.655x22 + 1.551x32
The parameters of Eqns (3)–(5) together with their t-ratio val-
ues and the ANOVA results are shown in Supplementary Material
Tables S1–S3. The significance of the models was checked by the
F-test. For TPC, the F-value (10.8) was higher than the tabulated
value for the 0.05 significance level; for oleuropein and hydroxyty-
rosol the developed models were significant as the F-values were
6.416 and 3.652, respectively.
Figure 2 illustrates the experimental and predicted values of
the developed models for TPC [Fig. 2(A)], oleuropein [Fig. 2(B)]
and hydroxytyrosol [Fig. 2(C)]. The R2 values between the model
predictions and experimental results were 0.84 for TPC, 0.89 for
oleuropein and 0.82 for hydroxytyrosol. However, R2 for tyrosol
was < 0.80 (0.77), and hence, this model was not taken into
consideration in this study.
The importance of the parameters was assessed by Student’s
t-distribution (see Tables S1–S3). For TPC, the most important
parameters were the quadratic parameter of temperature (𝛽 1 𝛽 1 ),
the linear effect of L : S ratio (𝛽 3 ), the quadratic parameter of
L : S (𝛽 3 𝛽 3 ) and the interaction parameter of the three variables
(𝛽 1 𝛽 2 𝛽 3 ). From the above parameters, only 𝛽 3 𝛽 3 a positive impact
had whereas all the rest had a negative impact. For oleuropein,
the most important parameters were the linear effect of H2 SO4 (𝛽 2 )
which had a negative impact and the linear effect of L : S ratio (𝛽 3 )
which had a positive impact. Regarding the hydroxytyrosol extrac-
460
eter: it affected positively the concentration of hydroxytyrosol pro-
duced from the hydrolytic procedure of oleuropein, whereas no
addition of H2 SO4 resulted in zero hydroxytyrosol in the extracts.
The model predictions of Eqn (3) for the examined area of TPC
(5)
are shown in Fig. 3 for X 1 (temperature) and X3 (L : S ratio) vari-
ables in the form of a 3D plot [Fig. 3(A)] and a contour plot
[Fig. 3(B)]. Optimum TPC extraction conditions (>85 mg GAE g –1
db) are between 32 and 38 ∘ C, and at a L : S ratio between
20 and 25.
Regarding the extraction of oleuropein, the model predictions
of Eqn (4) are illustrated in Fig. 4 for X 2 and X 3 variables in the
form of a 3D plot [Fig. 4(A)] and a contour plot [Fig. 4(B)]. The
optimum extraction conditions for oleuropein in the examined
area took place at maximum L : S ratio and minimum H2 SO4
addition, designating, on the one hand, the positive effect of the
L : S ratio, and on the other, the negative effect of H2 SO4 on the
extraction of this phenolic compound.
Validation of the developed models was carried out on the opti-
mum conditions for TPC and oleuropein extraction. The experi-
mental procedure was the same with the one followed on the CCD
runs. The values for X 1 , X 2 and X 3 for optimum TPC extraction were
31.9 ∘ C, 6.4% H2 SO4 (v/w) and 25.2 L : S (v/w), whereas those for
optimum oleuropein extraction were 40 ∘ C, 2.0% H2 SO4 (v/w) and
30 L : S (v/w). The obtained experimental result for TPC, at the opti-
mum conditions, was 86.4 mg GAE g –1 db, a result very close to
the model prediction of 86.2 mg GAE g –1 db. For oleuropein, the
model seemed to overpredict the experimental values on extreme
conditions (±1.6818 level) as the experimental run at the opti-
mum conditions was 43.1 mg g−1 db and the model prediction was

tion, it was positively affected by the added amount of H2 SO4 . The 56.9 mg g−1 db.

wileyonlinelibrary.com/jctb © 2019 Society of Chemical Industry J Chem Technol Biotechnol 2020; 95: 457–465
 10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Extraction of phenolic compounds from olive leaves www.soci.org

(A) 90 (A)
Model Predictions TPC

80
(mg GAE g–1 db)

70

60

50

40
40 50 60 70 80 90
Experimental TPC (mg GAE g–1 db)
(B) 50
Model Predictions oleuropein

40 (B)
40
(mg g–1 db)

50

65

80
85
30 38

70
55

75

85
36

60

75
20
34

80
Temperature (oC)

85
32
65
10

80
10 20 30 40 50
30 70

75

70
Experimental oleuropein (mg g–1 db)
28 75
(C) 80
10
Model Predictions hydroxytyrosol

26

60
55
75
8 24 70 65
65

70
50
(mg g–1 db)

60

22
6 45
65 60 40 5
3
55

20 55
4 10 12 14 16 18 20 22 24 26 28 30
L/S ratio (v/w)
2
Figure 3. Model predictions on the examined area for TPC with respect to
X 1 (temperature) and X 3 (L : S ratio) in 3D plot (A) and contour plot (B); the
0
coded value for H2 SO4 (X 2 ) was set to zero.
0 2 4 6 8 10
–1
Experimental hydroxytyrosol (mg g db)
content presented an AAI of 0.45. However, these extracts were
Figure 2. Experimental results and model predictions of the three outputs taken directly from the solid–liquid extraction process, and hence,
of the CCD, TPC (A), oleuropein (B) and hydroxytyrosol (C).
were not concentrated throughout evaporation. The antioxidant
activity of the samples is expected to be significantly higher once
the extracts are vaporized and concentrated.
Extraction kinetics on the optimum conditions for TPC Both kinetics were fitted with a logarithmic type of equation
and oleuropein
using linear regression analysis [see Eqns (6) and (7) for TPC and
Extraction kinetics also were studied for TPC and oleuropein oleuropein, respectively]. Both equations fitted well to experimen-
for the optimum extraction conditions indicated by the CCD tal results as R2 was 0.96 for TPC and 0.98 for oleuropein.
and results are shown in Fig. 5. Extraction kinetics of hydroxy-
( )
tyrosol and tyrosol were omitted due to their low amounts in TPC mg g−1 db = 10.125 ln (t) + 30.294 (6)
the extracts (<10 mg g−1 db). For TPC, high extraction content
(>78 mg GAE g –1 db) occurred during the first 90 min of extraction. ( )
After that time point, TPC increased very slowly. The maximum Oleuropein mg g−1 db = 7.7366 ln (t) –1.2464 (7)
extraction rate took place in the first 45 min where >90% of the
final TPC had been extracted. Oleuropein extraction occurred until
135 min where >95% of the final oleuropein had been extracted. DISCUSSION
The AAI results of the extracts at the optimum TPC and oleu- Olive oil is an important part of the Greek economy as it consitutes
ropein conditions showed weak antioxidant activity, as indicated 9% of the total agricultural production value in Greece, whereas
by the scale proposed previously.20 The extract with highest TPC the sector has been characterized as highly dynamic because
461

exhibited an AAI of 0.32, whereas that with the highest oleuropein Greece is the third largest oil producer in the world (after Spain

J Chem Technol Biotechnol 2020; 95: 457–465 © 2019 Society of Chemical Industry wileyonlinelibrary.com/jctb
 10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org GK Lamprou et al.

(A) (A)
100
90

TPC (mg GAE g–1 db)

80
70
60
50
40
30
20
10
0
0 50 100 150 200 250 300 350

Time (min)
(B)
50
45

Oleuropein (mg g–1 db)

40
(B)
30 35
35

30
40

28
25
25

20
26
30

15
24 10
5
20
L/S ratio (v/w)

35

22 0
15
40

0 50 100 150 200 250 300 350

25

20
30
Time (min)
18
Figure 5. Extraction kinetics of TPC (A) and oleuropein (B) at their optimum
35 20 extraction conditions.
10

16
25
14 30
12
20
10 25 15
2 3 4 5 6 7
Sulphuric acid % (v/w)
Figure 4. Model predictions on the examined area for oleuropein with
respect to X 2 (H2 SO4 ) and X 3 (L : S ratio) in 3D plot (A) and contour plot
(B); the coded value for temperature (X 1 ) was set to zero.
and Italy) with annual production of around 0.3 Mt, accounting for
0.4% of its GDP. Although consumption remains concentrated in
the producing countries, new markets are emerging worldwide
and are exhibiting a strong and increasing demand. Most olive
mills in Greece are smaller and less technologically advanced than
those in Spain, leading to higher olive oil production costs. In Italy,
even though the olive mills are of low capacity they are vertically
integrated with the olive farming stage as well as the distribution
stage. Valorization of olive mill by-products such as olive mill
wastes and olive leaves can increase the economic efficiency of
this food industry sector converting it to modern biorefineries
where, apart from olive oil, it also produces a range of valuable
products such as extracts rich in phenolic compounds, biofuels
and compost.28
The scope of this study was to identify the optimum values of
three important factors (temperature, amount of H2 SO4 and the
dilution ratio) for extracting the phenolic compounds contained
in olive leaves using a solvent extraction process. For this purpose,
a 33 central composite design was selected for the extraction
process so as to optimize the most important outputs namely TPC,
oleuropein and hydroxytyrosol. Apart from H2 SO4 , hydrochloric
462
15
shown) for the hydrolysis/acidification of extracts. These tests
5 showed that the performance of HCl was significantly lower than
10
that of H2 SO4 . On top of that, H2 SO4 is a very cheap chemical and
0
less toxic to the environment than HCl.
8 9 10
Applications for phenolic compounds have been researched
and applied primarily in the food and pharmaceutical industries,
but recently new applications of these compounds have been
introduced. In our study, the final extract is targeted for use as
a biopesticide in pest management operations. The advantages
of using these extracts for bulk purposes and not only in niche
(limited) markets include the relatively simple separation and
purification processes required to recover phenolic compounds at
high purities.
New extraction techniques have been examined in order to
achieve higher extraction efficiencies with lower environmental
impacts. These extraction techniques such as microwave, super-
critical fluid, pressurized liquid, fractionation by solid-phase and
dynamic ultrasound, have been used mostly at laboratory scales.
They have not been scaled-up to industry yet, mainly due to
their high operational and capital costs. This is mainly why this
study focuses on the development of a simple and cost-effective
methodology that can be easily implemented in existing olive
mills for extracting high added-value phenolic compounds con-
tained in olive leaves. For the proposed process, both capital and
operational cost are very low: there is no need for new capital
investment as olive mills can use their existing equipment (mix-
ers, decanters) and sulphuric acid is a very low-cost operational
chemical (∼0.2 US$ kg−1 ). Hence, the proposed process creates a
stimulus for the development of an integrated management and
waste valorization process from olive mills. The adoption of the
methodology, is expected to have a positive socioeconomic effect

acid (HCl) also was tested in preliminary experiments (results not in areas with a high dependency from olive sector. The valorization

wileyonlinelibrary.com/jctb © 2019 Society of Chemical Industry J Chem Technol Biotechnol 2020; 95: 457–465
 10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Extraction of phenolic compounds from olive leaves www.soci.org

Table 3. Results from the literature on the extraction of TPC and oleuropein from olive leaves

Solvent Cultivar TPC (mg GAE g –1 db) Oleuropein (mg g –1 db) Reference

Methanol Olea europaea var. sylvestris 20.60 ND 22

Methanol Chemlali 14.40 ND 27
Methanol 25% Chemlali ND 24.40 25
Methanol 20% Chemlali 15.55 ND 21
Methanol 20% NDa 26.80 ND 23
Methanol 40% NDa 78.90 ND 23
Methanol 60% NDa 79.20 ND 23
Methanol 80% NDa 61.90 ND 23
Methanol 80% Koroneiki ND 0.50 9
Methanol 80% Chemlali 13.80 ND 9
Methanol 80% Arbequina 60.64 18.51 26
Methanol 80% Jlott ND 9.2 29
Ethanol Olea europaea var. sylvestris 27.30 ND 22
Ethanol 20% NDa 46.00 ND 23
Ethanol 40% NDa 66.30 ND 23
Ethanol 50% Olea europaea var. sylvestris 24.80 ND 22
Ethanol 60% NDa 66.70 ND 23
Ethanol 70% Meski 110.03 52.63 24
Chetoui 94.03 57.24
Ethanol 70% Sevillane 56.75 30.76 9
Ethanol 80% NDa 78.10 ND 23
Acetone 20% NDa 58.20 ND 23
Acetone 40% NDa 71.50 ND 23
Acetone 60% NDa 79.80 ND 23
Acetone 80% NDa 84.40 ND 23
Acetone: ethanol:H2 O (6:2:2) NDa 60.66 ND 23
Acetone: ethanol:H2 O (4:4:2) NDa 89.80 ND 23
Glycerol 9.3% (80 ∘ C) Koroneiki 51.91 ND 30
Ethyl acetate Olea europaea var. sylvestris 13.50 ND 22
n-Propanol Olea europaea var. sylvestris 13.70 ND 22
Isopropanol Olea europaea var. sylvestris 13.50 ND 22
Water acidified with HCl (pH 3, at 60 ∘ C for 4 h) Sarpol e Zahab ND 6.01 31
Kermanshah ND 6.40
Kerman ND 10.20
Shiraz ND 13.00
Paveh ND 7.40
Amol ND 6.50
Behshahr ND 9.60
Roodbar ND 9.00
Water acidified with H2 SO4 (31.9 ∘ C, 6.4% Koroneiki 86.40 ND This study
H2 SO4 , 25.2 L : S (v/w))
Water acidified with H2 SO4 (40.9 ∘ C, 2.0% Koroneiki ND 43.10 This study
H2 SO4 , 30 L : S (v/w))
a The variety is not defined. The orchard is located in area of Safita/Tartous in Syria.

of by-products will offer an extra income to olive mill owners and
olive tree farmers. In this study, water was selected as solvent
instead of ethanol or acetone in order to reduce the cost of the pro-
cess and eliminate the risk of introducing toxic compounds in the
final extract. Our final aim is to use the olive leave extracts without
any further processing (i.e. evaporation) as biopesticides. The latter
are usually dissolved in water by the end-user.
The developed method achieved a high extraction yield of
phenolic compounds reaching 85% of the initial TPC. The latter
was measured by executing repeated extractions using the same
raw material. Many studies in the literature have examined the
using readily available agro-industrial alcohols (pure or mixed with
water). However, methanol would have negative effects in the
final extract owing to its toxicity, thus limiting many potential
applications, and ethanol would increase operational due to its
high value.
In Table 3, we compare the results from the literature with
those obtained in this study for the extraction of phenolic
compounds from olive leaves. According to the literature, the
TPC yield using a solid–liquid extraction process ranges from
13.5 to 110 mg GAE g –1 db whereas the concentration of oleu-
ropein extracted from olive leaves ranges from 0.5 to 57 mg g−1
463

solid–liquid extraction of phenolic compounds from olive leaves db. Maximum TPC (110.03 mg GAE g –1 db) and oleuropein

J Chem Technol Biotechnol 2020; 95: 457–465 © 2019 Society of Chemical Industry wileyonlinelibrary.com/jctb

www.soci.org
(57.24 mg g−1 db) extraction have been achieved when an aque-
ous solvent with 70% ethanol was used.24 Extracts with high TPC
values (>78 mg GAE g –1 db) also were achieved using diluted
methanol at methyl alcohol (MeOH) : water ratios of 40:60 or
60:40.23 Pure methanol or aqueous solutions with <40% MeOH
generate extracts with substantial lower TPC (<30 mg GAE g –1
db).21,22,23,25,27 Similar TPC results were observed when ethanol
was used as pure solvent.22 Acetone/water solutions of 20% to
80% acetone had a very high effectiveness of TPC extraction
reaching a maximum of 84.4 mg GAE g –1 db at 80% acetone.23
The same authors also used a mix of acetone: ethanol: H2 O (4:4:2)
and obtained 89.8 mg GAE g –1 db.23 Results obtained in this study
are very close to the highest values published in the literature both
for the TPC (86.4 mg GAE g –1 db) and oleuropein (43.1 mg g−1 db).
On top of that, the developed process employs low-value H2 SO4 in
small amounts, offering through its simplicity the right conditions
to scale this process up in industrial applications integrated within
olive mill facilities.
CONCLUSIONS
In the present study, a new cost-effective process for the extraction
of phenolic compounds from olive leaves was examined. The resul-
tant extract, after hydrolysis, contained an optimum TPC value
of 86.4 mg GAE g –1 db and an optimum amount of oleuropein of
43.2 mg g−1 db. Moreover, ≤8 mg of hydroxytyrosol per gram dry
olive leaves can be produced from the hydrolysis of oleuropein
when elevated amounts of H2 SO4 are used in the hydrolysis
process. New applications for these phenolic compound-rich
extracts are being explored and applied by the research com-
munity, conferring this food waste with higher value than
ever before.
ACKNOWLEDGEMENTS
This research is co-financed by Greece and the European Union
(European Social Fund- ESF) through the Operational Programme
‘Human Resources Development, Education and Lifelong Learn-
ing’ in the context of the project ‘Strengthening Human Resources
Research Potential via Post-Doc Research’ (MIS-5001552), imple-
mented by the State Scholarships Foundation (IKY).
ANOVA tables regarding the results of the CCD for oleu-
ropein, hydroxytyrosol and TPC extraction are available as
e-supplementary data that can be found in the e-version of
this paper online.
Supporting Information
Supporting information may be found in the online version of this
article.
REFERENCES
1 Pandey KB and Rizvi SI, Plant polyphenols as dietary antioxidants
in human health and disease. Oxid Med Cell Longev 2:270–278
(2009).
2 Talhaoui N, Taamalli A, Gómez-Caravaca AM, Fernández-Gutiérrez A
and Segura-Carretero A, Phenolic compounds in olive leaves: analyt-
ical determination, biotic and abiotic influence, and health benefits.
Food Res Int 77:92–108 (2015).
3 European Food Safety Authority (EFSA) Panel on Dietetic Products
Nutrition and Allergies (NDA), Scientific opinion on the substan-
tiation of health claims related to polyphenols in olive and pro-
tection of LDL particles from oxidative damage. EFSA J 9:1–25
464
(2011).
wileyonlinelibrary.com/jctb © 2019 Society of Chemical Industry
10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GK Lamprou et al.
4 Moudache M, Colon M, Nerín C and Zaidi F, Phenolic content and
antioxidant activity of olive by-products and antioxidant film con-
taining olive leaf extract. Food Chem 212:521–527 (2016).
5 Daglia M, Polyphenols as antimicrobial agents. Curr Opin Biotechnol
23:174–181 (2012).
6 Turck D, Bresson J, Burlingame B, Dean T, Fairweather-Tait S,
Heinonen M et al., Safety of hydroxytyrosol as a novel food pursuant
to Regulation (EC) No 258/97. EFSA J 15:1–23 (2017).
7 Dai J and Mumper RJ, Plant phenolics: extraction, analysis and their
antioxidant and anticancer properties. Molecules 15:7313–7352
(2010).
8 El SN and Karakaya S, Olive tree (Olea europaea) leaves: poten-
tial beneficial effects on human health. Nutr Rev 67:632–638
(2009).
9 Souilem S, Fki I, Kobayashi I, Khalid N, Neves MA, Isoda H et al., Emerging
technologies for Recovery of value-added components from olive
leaves and their applications in food/feed industries. Food Bioprocess
Technol 10:229–248 (2017).
10 Vlyssides AG, Lamprou GK and Vlysidis A, Industrial case studies on the
detoxificaton of OMWW using Fenton oxidation process followed by
biological processes for energy and compost production, in Olive
Mill Waste, ed. by, ed. by Galanakis CM. Elsevier, Amsterdam, pp.
119–138 (2017).
11 Bampalioutas K, Vlysidis A, Lyberatos G and Vlyssides A, Detoxifica-
tion and methane production kinetics from three-phase olive mill
wastewater using Fenton’s reagent followed by anaerobic digestion.
J Chem Technol Biotechnol 94:265–275 (2019).
12 Romero-García JM, Niño L, Martínez-Patiño C, Álvarez C, Castro E and
Negro MJ, Biorefinery based on olive biomass. State of the art and
future trends. Bioresour Technol 159:421–432 (2014).
13 Mitsopoulos G, Hagidimitriou M, Papageorgiou V and Komaitis M, Total
phenolic content, phenolic profile and antioxidant activity in leaves
and drupes of Greek olive cultivars. Acta Hortic 924:425–430 (2011).
14 Rosati A, Cafiero C, Paoletti A, Alfei B, Caporali S, Casciani L et al., Effect
of agronomical practices on carpology, fruit and oil composition,
and oil sensory properties, in olive (Olea europaea L.). Food Chem
159:236–243 (2014).
15 Rahmanian N, Jafari SM and Wani TA, Bioactive profile, dehydration,
extraction and application of the bioactive components of olive
leaves. Trends Food Sci Technol 42:150–172 (2015).
16 Nikolaivits E, Termentzi A, Skaltsounis AL, Fokialakis N and Topakas E,
Enzymatic tailoring of oleuropein from Olea europaea leaves and
product identification by HRMS/MS spectrometry. J Biotechnol
253:48–54 (2017).
17 Agalias A, Magiatis P, Skaltsounis A-LL, Mikros E, Tsarbopoulos A,
Gikas E et al., A new process for the management of olive oil mill
waste water and recovery of natural antioxidants. J Agric Food Chem
55:2671–2676 (2007).
18 De Leonardis A, Aretini A, Alfano G, Macciola V, Ranalli G, Jemai H
et al., Isolation of a hydroxytyrosol-rich extract from olive leaves
(Olea Europaea L.) and evaluation of its antioxidant properties and
bioactivity. Eur Food Res Technol 226:653–659 (2008).
19 Alexandri M, Papapostolou H, Vlysidis A, Gardeli C, Komaitis M,
Papanikolaou S et al., Extraction of phenolic compounds and suc-
cinic acid production from spent sulphite liquor. J Chem Technol
Biotechnol 91:2751–2760 (2016).
20 Scherer R and Godoy HT, Antioxidant activity index (AAI) by the
2,2-diphenyl-1-picrylhydrazyl method. Food Chem 112:654–658
(2009).
21 Bouallagui Z, Han J, Isoda H and Sayadi S, Hydroxytyrosol rich extract
from olive leaves modulates cell cycle progression in MCF-7 human
breast cancer cells. Food Chem Toxicol 49:179–184 (2011).
22 Lafka T-I, Lazou A, Sinanoglou V and Lazos E, Phenolic extracts from
wild olive leaves and their potential as edible oils antioxidants. Foods
2:18–31 (2013).
23 Wissam Z, Ali A and Rama H, Optimization of extraction conditions for
the recovery of phenolic compounds and antioxidants from Syrian
olive leaves. J Pharmacogn Phytochem 5:390–394 (2016).
24 Ben Salah M and Abdelmelek H, Study of phenolic composition and
biological activities assessment of olive leaves from different vari-
eties grown in Tunisia. Med Chem (Los Angeles) 2:107–111 (2012).
25 Jemai H, Feki AEL, Sayadi S, El Feki A and Sayadi S, Antidiabetic and
antioxidant effects of hydroxytyrosol and oleuropein from olive
leaves in alloxan-diabetic rats. J Agric Food Chem 57:8798–8804
(2009).
J Chem Technol Biotechnol 2020; 95: 457–465

Extraction of phenolic compounds from olive leaves
26 Talhaoui N, Gómez-Caravaca AM, León L, De la Rosa R,
Segura-Carretero A and Fernández-Gutiérrez A, Determination of
phenolic compounds of ‘Sikitita’ olive leaves by HPLC-DAD-TOF-MS.
Comparison with its parents ‘Arbequina’ and ‘Picual’ olive leaves.
LWT - Food Sci Technol 58:28–34 (2014).
27 Boudhrioua N, Bahloul N, Ben Slimen I, Kechaou N. Comparison
on the total phenol contents and the color of fresh and infrared
dried olive leaves. Industrial Crops and Products 29:412–419
(2009).
28 Galanakis CM, Olive Mill Waste: Recent Advances for the Sustainable
Management. Elsevier - Academic Press, (2017).
J Chem Technol Biotechnol 2020; 95: 457–465 © 2019 Society of Chemical Industry
10974660, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jctb.6049 by Ege Universitesi, Wiley Online Library on [03/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org
29 Tayoub G, Sulaiman H, Hassan AH and Alorfi M, Determination of
oleuropein in leaves and fruits of some Syrian olive varieties. Int J Med
Aromat Plants 2:428–433 (2012).
30 Apostolakis A, Grigorakis S and Makris DP, Optimisation and compara-
tive kinetics study of polyphenol extraction from olive leaves (Olea
europaea) using heated water/glycerol mixtures. Sep Purif Technol
28:89–95 (2014).
31 Ansari M, Kazemipour M and Fathi S, Development of a simple green
extraction procedure and HPLC method for determination of oleu-
ropein in olive leaf extract applied to a multi-source comparative
study. J Iran Chem Soc 8:38–47 (2011).
465
wileyonlinelibrary.com/jctb