Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives determined by measuring malondialdehyde in rat brain

Article abstract of DOI:10.1111/jphp.12807

Objectives: The purpose of this study was the evaluation of the antioxidant activity of natural and semisynthetic polyphenol derivatives from Olea europea L., by assessing malondialdehyde (MDA), an important marker of oxidative stress. Methods: Polyphenol as hydroxytyrosol, oleuropein, oleuropein aglycone as mix of four tautomeric forms and their respective acetyl-derivatives were obtained from olive leaves using semisynthetic protocols. These compounds were administered intraperitoneally to Wistar rats treated with paraquat, an herbicide which is able to cause oxidative stress after central administration. Malondialdehyde was derivatized with 2,4-dinitrophenylhydrazine to produce hydrazone that was purified by solid-phase extraction. Using high-performance liquid chromatography coupled with a diode array, free and total MDA was measured on homogenate rat brain as marker of lipid peroxidation. The analytical method was fully validated and showed linearity in the tested concentration range, with detection limit of 5 ng/ml. Recovery ranged from 94.1 to 105.8%. Key findings: Both natural and semisynthetic polyphenol derivatives from a natural source as olive leaves were able to reduce MDA detection. The more lipophilic acetyl-derivatives showed an antioxidant activity greater than parent compounds. This potency seems to put in evidence a strict correlation between lipophilicity and bioavailability.

Full text of DOI:10.1111/jphp.12807

Research Paper
Antioxidant activity of oleuropein and semisynthetic
acetyl-derivatives determined by measuring
malondialdehyde in rat brain
a
b
a
a
Milena Rizzo
, Domenica Ventrice , Francesca Giannetto
, Salvatore Cirinn aꢀ ,
a
c
c
c
Natale Alfredo Santagati , Antonio Procopio , Vincenzo Mollace and Carolina Muscoli
a
b
c
Department of Drug Sciences, University of Catania, Catania, Italy, ARPACAL, Catanzaro, Italy and Department of Health Sciences, University of
Magna Græcia, Catanzaro, Italy
Keywords
Abstract
antioxidant activity; oleuropein and
semisynthetic derivatives; malondialdehyde
Objectives The purpose of this study was the evaluation of the antioxidant activ-
ity of natural and semisynthetic polyphenol derivatives from Olea europea L., by
assessing malondialdehyde (MDA), an important marker of oxidative stress.
Methods Polyphenol as hydroxytyrosol, oleuropein, oleuropein aglycone as mix
of four tautomeric forms and their respective acetyl-derivatives were obtained
from olive leaves using semisynthetic protocols. These compounds were adminis-
tered intraperitoneally to Wistar rats treated with paraquat, an herbicide which is
able to cause oxidative stress after central administration. Malondialdehyde
was derivatized with 2,4-dinitrophenylhydrazine to produce hydrazone that was
purified by solid-phase extraction. Using high-performance liquid chromatogra-
phy coupled with a diode array, free and total MDA was measured on homoge-
nate rat brain as marker of lipid peroxidation. The analytical method was fully
validated and showed linearity in the tested concentration range, with detection
limit of 5 ng/ml. Recovery ranged from 94.1 to 105.8%.
Correspondence
Milena Rizzo, Department of Drug Science,
University of Catania, Viale Andrea Doria, 6
9
5125 Catania, Italy.
E-mail: milena.rizzo@unict.it
Received February 3, 2017
Accepted July 26, 2017
doi: 10.1111/jphp.12807
Key findings Both natural and semisynthetic polyphenol derivatives from a nat-
ural source as olive leaves were able to reduce MDA detection. The more lipophi-
lic acetyl-derivatives showed an antioxidant activity greater than parent
compounds. This potency seems to put in evidence a strict correlation between
lipophilicity and bioavailability.
that they exhibit good radical scavenging abilities and
Introduction
[
8]
superoxide dismutase-like activity. Indeed, olive leaves
have been used widely in traditional remedies in European
and Mediterranean countries; they have been used in the
human diet as extracts, herbal teas and powders. In addi-
tion, olive leaves (an agricultural waste product) have great
potential as a source of a natural antioxidant, that is Oleu-
ropein (Ole) that can constitute up to 6–9% of the dry mat-
Epidemiological studies have shown a relationship between
the Mediterranean diet and a lowered incidence of patholo-
gies such as cancer, atherosclerosis, cardiovascular and neu-
[1,2]
rodegenerative diseases.
protective action is played by the presence of olive oil.
A key role in this diet’s
[
3–5]
Olive extracts are a natural source of polyphenols, widely
considered to be potentially beneficial for health. Many
studies have revealed their capacity to scavenge reactive
species, and human intervention studies have shown that
olive polyphenols decrease the levels of oxidized low-den-
sity lipoproteins in plasma and positively affect several
[
9]
ter derived from olive trees. Simple treatments have been
evaluated as procedures to recover natural antioxidants
[
10]
from olive leaves.
Ole belongs to a specific group of coumarin-like com-
pounds, the secoiridoids, which are usually found only in
plants belonging to the Oleaceae family.
[6,7]
biomarkers of oxidative damage. A more recent study
assessed the antioxidant activity of both individual and
combined phenolics in Olea europaea L. leaf extract, finding
Ole is a glucoside ester of hydroxytyrosol (HT) and
elenolic acid. Secoiridoids, in aglyconic forms, arise from
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
1

Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Milena Rizzo et al.
glycosides in olive fruits by hydrolysis of endogenous b-glu-
cosidases during crushing and malaxation. These newly
formed substances, which have amphiphilic characteristics,
are partitioned between the oily layer and the vegetation
water and are more concentrated in the latter fraction
because of their polar functional groups.
Experimental
Chemicals and standards
Acetonitrile (ACN), acetone (C H O), benzoic acid, cate-
6
3
chol, chloral hydrate (C H Cl O ), dichloromethane
3 2
2
3
Carrasco-Pancorbo et al. determined the antioxidant
activity of several single phenolic compounds in virgin olive
oil and showed that HT and Ole aglycone were the stron-
(CH Cl ), 2,4-dinitrophenylhydrazine (DNPH), phosphate-
2 2
buffered saline (PBS), methanol (MeOH), naphthalene,
naphthol, paraquat dichloride (PQ), perchloric acid
(HClO4), resorcinol, sodium dihydrogen phosphate,
sodium chloride (NaCl), sodium hydroxide (NaOH), sulfu-
ric acid (H SO ), trifluoroacetic acid (CF COOH), 1,1,3,3-
[11]
gest in terms of antioxidant capacity.
A recent review
summarizes the findings on Ole aglycone’s beneficial effects
against neuro-degeneration and other peripheral inflamma-
2
4
3
[12]
tory and degenerative diseases.
That activity could
tetraethoxypropane (TEP) were purchased from Sigma-
Aldrich Co. (Milan, Italy). All reagents were of analytical
grade, unless stated otherwise and the organic solvents were
HPLC grade. Water used for the preparation of a mobile
phase and chemical reagents was prepared using an Elix TM
water purification system (Millipore, Toronto, Canada).
result, at least in part, in a remarkable improvement in the
pathological signs arising from stress conditions such as
oxidative stress. The relative antioxidant potency of indi-
vidual olive oil phenols may vary depending on their
bioavailability. Several studies, carried out with human and
animal models, have shown good bioavailability of olive oil
[
13,14]
phenols.
Ole is rapidly absorbed after oral administra-
Oleuropein and its semisynthetic derivatives
tion, with maximum plasma concentration occurring 2 h
after administration. Moreover, an important step in the
metabolism of olive oil phenolics, that is Ole-glycoside, Ole
and ligistroside–aglycones, is their formation into HT or
tyrosol. These compounds are rapidly distributed and
excreted in urine mainly as glucuronides or in very low
concentrations as free forms. Indeed, HT is the most
important metabolite.
Ole was extracted from Coratina olive cultivar leaves, using a
conventional extraction procedure. The olive leaves were
dried for 48 h at 50 °C, milled and refluxed with water
(100 g/l) at 100 °C for 8 h. The leaves were filtered, and the
solution was dried under reduced pressure. The mixture was
treated with C H O and filtered to remove solids; the solu-
6
3
tion was evaporated under reduced pressure to obtain the
crude product. Ole was separated by means of liquid chro-
matography on a Supelco Versa–Flash station equipped with
To enhance bioavailability, various lipophilic com-
pounds have been synthesized using a simple and environ-
mentally friendly semisynthetic protocol by chemical
manipulation of complex natural substances. Procopio
et al. reported an international patent for a chemical-cata-
lytic method for the peracetylation of Ole and its deriva-
a silica cartridge and eluted with a mixture of CH Cl /MeOH
2 2
(8 : 2 v/v) as mobile phase. HT, that is 2-(3,4-dihydroxyphe-
nyl)ethanol, that occurs as simple phenol or esterified with
elenolic acid to form Ole, was synthesized as reported in the
[20]
[
15]
tives.
acetal linkage hydrolysis mimicking natural glycosidase
The aglycone was easily obtained from Ole by
literature.
Ole aglycone was obtained through controlled
hydrolysis as a mixture of four tautomeric forms (OleAa-d):
the enolic and dialdehydic forms, OleAa and OleAb, respec-
tively, as well as the ring-closed species OleAc and the
[16]
enzyme action, and it has been studied in different
experimental animal models, showing interesting results as
[17,18]
[16]
an anti-inflammatory agent.
Synthesis of peracety-
hydrated aldehydic derivative OleAd.
Then, acetylation of
Ole, OleAa-d and HT was performed to obtain more lipo-
lated derivatives was performed to test their anti-inflamma-
tory activity, and they were then successfully applied to
philic acetyl-derivatives, that is AcOle, AcOleAa-d and AcHT,
[15]
respectively (Figure 1a and 1b).
[16,19]
study breast and thyroid cancer models.
The purpose of this study was the evaluation of the
antioxidant activity of both natural and semisynthetic
polyphenol derived from Olea europea L. leaves. Then, the
more lipophilic aglycone and acetyl-derivatives were pre-
pared to determine a correlation between lipophilicity and
bioavailability. An in-vivo rat model was chosen, and
malondialdehyde (MDA) was determined as marker of
oxidative stress induced by paraquat. A newly developed
extraction procedure of hydrazone derivative from homo-
genate brain and high-performance liquid chromatography
Determination of lipophilicity
The lipophilicity of the studied derivatives was measured
by determining the partition coefficient (LogP). The experi-
mental LogP was determined by reversed-phase liquid
[16]
chromatography (RP-HPLC), as previously described.
Stock solutions containing 10 lg/ml of single polyphenols
were prepared by dissolving them in mobile-phase solution
(30% water/CF COOH, pH = 2.4, 70% methanol). Multi-
3
(
HPLC) was fully validated.
ple injections of single standards were performed to define
2
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Milena Rizzo et al.
Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Figure 1 (a) Chemical structures of Ole, HT and their respective peracetylated derivatives AcOle and AcHT. (b) Chemical structures of oleuropein
hemiacetal aglycone as a mixture of four tautomeric forms OleAa-d, that is three isomers (a-c), hydrated aldehydic form (d). (c) Chemical struc-
tures of acetylated derivatives of oleuropein hemiacetal aglycone as a mix of four tautomeric forms AcOleAa-d.
individual retention times and their reproducibility, as well
as the stability of these selected compounds. Five reference
compounds (catechol, resorcinol, benzoic acid, naphthol
and naphthalene) with sufficiently different retention times
stress after central administration (icv) in rats. Male Wistar
rats (280–300 g; Charles River, Milan, Italy) were housed
three per cage in humidity (60 ꢀ 5%) and temperature
(22 ꢀ 2 °C)-controlled environment and were allowed free
access to food and water until the time of the experiments.
The animals were kept in a 12-h light/12-h dark cycle (lights
on 7.00 a.m.–7.00 p.m., off 7.00 p.m.–7.00 a.m.). Animal
care was in compliance with Italian regulations on protec-
tion of animals used for experimental and other scientific
purposes (DM 116192) as well as with EEC regulations (OJ
of ECL 358/1 12/18/198 6). Before the experiment, rats were
anaesthetized with C H Cl O (400 mg/kg), intraperi-
0
were mixed. Capacity factor k was calculated for each com-
0
pound. LogK values were correlated with known LogP val-
ues through a linear regression analysis to obtain a
correlation curve. Moreover, experimental data were com-
pared to theoretical data to determine how peracetylation
improved the lipophilic character of polyphenols. The the-
oretical LogP values were obtained using two different
methods: one based on established chemical interactions
2
3
3 2
(
Switzerland) and the other based on group contributions
cLogP calculated by Actelion Cheminformatics, Allschwil,
toneally (ip), and positioned in a stereotaxic frame (D. Kopf
Instruments). A stainless steel guide cannula (25 gauges)
was then implanted unilaterally into the Substantia Nigra
under stereotaxic guidance and secured to the skull with
dental acrylic. The rats were allowed a one-week recovery
period after cannulae implantation. For testing, the animals
were placed in individual transparent Perspex cages and
allowed to acclimatize to the new environment for 15 min.
The rats were randomly divided into eight groups each
(miLogP calculated by Molinspiration Cheminformatics,
Bratislava, Slovak Republic).
[21,22]
Drugs administration
The antioxidant activity of the studied derivatives was evalu-
ated using the herbicide PQ, which is able to cause oxidative
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
3

Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Milena Rizzo et al.
comprising six animals treated using saline, PQ alone and
PQ plus whichever polyphenol derivative was being studied,
that is Ole, AcOle, OleAa-d, AcOleAa-d, HT and AcHT,
respectively. Infusion of PQ (150 lg/kg, icv) or vehicle
dissolved in 100 ml of deionized water to give a 1 mM stock
solution. MDA was prepared by hydrolysis of 1 ml TEP
stock solution in 50 ml H SO 1% and stirred for 2 h at
2
4
room temperature. Then, MDA derivatization reaction was
carried out for 30 min at room temperature using DNPH,
as shown in Figure 3. In detail, 25 ll of DNPH 5 mM (pre-
(
0.9% NaCl) was carried out by means of a 1-ml Hamilton
syringe connected by a Teflon tube to an injection cannulae
and injected in a total volume of 1 ll/min. The microinjec-
tion was considered successful if, after removal of the
microinjector, a liquid flow could be induced by pushing the
plunger therefore indicating that the injector was not
clogged. Polyphenols were given (10 lmol/kg, ip), 15 min
before focal injection of PQ. The dose and route of polyphe-
nol administration used here to reduce joint injury were
pared using 2.9 mg of DNPH in 3 ml of HClO 35%) was
4
added to 250 ll of the resulting MDA standard solution
(20 nmol/ml). All reactions were protected from exposure
to light. The resulting derivatized MDA was a 2,4-dinitro-
phenylhydrazone (HDZ) insoluble in aqueous medium. It
was filtered, dried, weighted and used as a standard to vali-
date the method by determining accuracy, precision,
extraction recovery and stability. Standard stock solution of
HDZ was prepared in ACN (10 lg/ml). Then, as a control
medium, TEP hydrolysis and MDA derivatization reac-
tionl were performed in PBS alone, whereas for method
validation, brain tissue derived from untreated rats was
used as homogenate PBS (1 : 20 p/v). In detail, 500 ll of
TEP solution was added to 500 ll of buffer or homogenate
[17,18]
chosen on the basis of previous studies.
Animals were
sacrificed by guillotine 4 h after treatment. Brains were col-
lected, immediately frozen in liquid nitrogen and kept at
ꢁ
80 °C until use. Each of the brains (from both treated and
control rats) was accurately weighted and homogenized in
PBS added to the brain in a ratio of 1 : 20 p/v (g of bio-
mass/ml of buffer), and the process was carried out in an
ice-bath for 3 cycles of 5 min each. The homogenate under-
went refrigerated centrifugation (14 000g for 15 min); the
supernatant was withdrawn, placed in Eppendorf test tubes,
each containing 500 ll of extracting phase and then cen-
trifuged at 8000g for 10 min. The supernatant was collected
and kept for estimation of endogenous MDA.
sample acidified with 500 ll of HClO 35%. The samples
4
were vortexed and then centrifuged. The supernatant was
collected and added to known amounts of DNPH for
30 min under stirring at 25 °C, to obtain HDZ. Homoge-
nate tissue derived from treated rats underwent different
preliminary procedures to determine free and total (free
and protein bound) MDA. Free MDA was prepared by acid
deproteinization, whereas an alkaline hydrolysis was chosen
to determine total MDA.
Validation
The method was validated by multiple analyses of spiked
pooled brain homogenate standards, with regard to linear-
ity, limit of detection (LOD) and quantification (LOQ),
precision, accuracy and extraction efficiency.
Free MDA
Homogenate rat brain samples (500 ll) and HClO 35%
4
(
500 ll) were mixed and the mixture vortexed to precipi-
tate protein. After centrifugation at 14 000g for 10 min,
clear supernatant was transferred to a 10-ml glass tube with
a conical glass stopper and added to DNPH solution
(100 ll, 5 mM) then mixed and incubated for 30 min at
25 °C in the dark.
MDA preparation, derivatization and
determination
For method validation, MDA was obtained by hydrolysis of
TEP, as reported in Figure 2. In detail, 25 ll TEP was
Figure 2 MDA obtained by acidic hydrolysis of TEP.
Figure 3 Derivatization process of endogenous and exogenous MDA with DNPH to obtain 2,4-dinitrophenylhydrazone(HDZ).
4
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Milena Rizzo et al.
Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Total MDA
each group of three rats. Comparisons of free and total
MDA content between the control group, the group treated
with paraquat and the groups treated with paraquat plus
each of the polyphenol derivatives studied were made using
one-way analysis of variance (ANOVA) with Tukey’s test
for post-hoc comparison. Differences were considered sig-
nificant at P < 0.05. Correlation analysis was performed
using Pearson’s test. All computations were performed
using Statgraphic Plus version 2.1 (Statistical Graphics
Corp. Rockville, MD).
To determine total MDA, an alkaline hydrolysis of the pro-
tein binding was performed on the bound MDA fraction:
50 ll of 2 M aqueous NaOH was added to 500 ll of homo-
genate brain rat sample and incubated in a 60 °C water
bath for 30 min., and then, the hydrolysed samples were
acidified with 100 ll of HClO 35%. After centrifugation at
4
1
1
4 000g for 10 min, the supernatant was transferred to a
0-ml glass tube with a conical glass stopper and added to
DNPH solution (100 ll, 5 mM), then mixed and incubated
for 30 min at 25 °C in the dark.
Results
Extraction
Experimental and calculated LogP
determination
Brains derived from untreated and treated rats underwent the
same extraction procedures using solid-phase extraction
Experimental LogP values were determined by RP-HPLC.
To calibrate the system, a suite of well-defined organic
compounds with known LogK’ values were used as stan-
dards. Then, LogP values of each studied polyphenol were
(
SPE). Conditioned cartridges, based on a polymeric matrix
with a high hydrophilic–lipophilic balance (HLB Oasis SPE
ml, 30 mg; Waters-Milford, MA, U.S.A.), and a LiChrolut
1
extraction unit (Merck) were chosen to isolate HDZ from
biological samples. Samples (1 ml) were applied to condi-
tioned cartridges and washed with 1 ml of water containing
MeOH 5%; then, analytes were eluted twice using 1 ml of
MeOH. For all dry steps, pressure was maintained at 39.9 kPa
for 3 min. The eluate was dried under a nitrogen stream at
0
found by correlation with LogK through a linear regression
(
LogP= a LogK’ + b) with a regression coefficient of 0.99.
Moreover, a comparison between the experimental and cal-
culated LogP was performed, as reported in Table 1.
All of the acetylated entities proved to be much more
lipophilic than the corresponding starting species, and a
good correlation was registered between the experimental
and calculated LogP values.
45 °C, and the residue was dissolved in 200 ll of mobile
phase. The extract underwent chromatographic separation.
The spiked brain homogenate samples were prepared to build
standard curves and were refrigerated at ꢁ20 °C until assay.
Chromatography
Representative chromatograms of homogenate rat brain
samples after SPE extraction are reported. An acid solution
of derivatizing agent DNPH and an organic solution of
derivatization product HDZ were injected into the column
and identified by their relative retention times:
Chromatography
A Jasco PU 1580 pump and LG 1580-02 ternary unit
(
Tokyo, Japan) with a 100-ll loop injection valve were
used. The chromatographic system was used with a Jasco
MD-1510 diode-array detector (DAD) (Tokyo, Japan),
working at 310 nm (the maximum absorption wavelength
of the HDZ). Data were processed using Borwin chro-
matography software (version 1.21) from Jasco (Tokyo,
Japan). A mixture of acidified water and ACN (45 : 55 v/v)
Table 1 Comparison between the experimental and calculated LogP
of oleuropein (Ole), acetylated oleuropein (AcOle), oleuropein agly-
cone as a mixture of tautomeric forms (OleAa-d), acetylated oleu-
ropein aglycone as
a mixture of tautomeric forms (AcOleAa-d),
was used as mobile phase, and CF COOH was chosen to
3
hydroxytyrosol (HT), acetylated hydroxytyrosol (AcHT)
reach pH = 2.2. It was delivered at a flow rate of 1 ml/min
through a Nucleosil 100-5 RP/18 (25 cm 9 4.6 cm, 4 lm)
reverse-phase column (Agilent, USA), with a C18 guard
column (4.5 cm 9 0.46 cm). A Gastorr GF 103 (Jones
Chromatography, Colorado, USA) block heater was utilized
to maintain the analytical column at 25 °C.
Calculated LogP
cLogP
Compounds
Experimental LogP
LogK’
miLogP
Ole
0.351
2.611
ꢁ0.37
2.050
1.430
2.854
1.100
2.050
ꢁ0.365
1.672
1.520
1.620
0.516
0.826
AcOle
OleAa-d
AcOleAa-d
HT
a
1.770
a
2.397
0.890
1.704
Statistical analysis
AcHT
a
Results are presented as the mean ꢀ standard deviation
The average value of the set of peaks corresponding to the complex
mixture was considered.
(
SD) and coefficient of variation percentage (CV %) within
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
5

Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Milena Rizzo et al.
3
.37 ꢀ 0.35 min for DNPH and 5.64 ꢀ 0.42 for HDZ,
HDZ (4.582 lg/ml) corresponding to free MDA deter-
mined in rats treated with PQ and pretreated with Ole; Fig-
ure 5c shows the measured concentration of HDZ
(1.796 lg/ml) corresponding to free MDA determined in
rats treated with PQ and pretreated with AcHT. All of the
chromatograms reported a peak corresponding to DNPH;
in fact, it was always added in excess to guarantee the com-
plete reaction of the unknown concentration of endoge-
nous MDA, formed in vivo in response to induced
oxidative stress.
respectively. The analytes were determined using a DAD set
at 310 nm. Figure 4 reports chromatograms of drug-free
rat samples. In detail, Figure 4a shows the chromatogram
without interfering peaks corresponding to the retention
times of the studied compounds, which could have affected
the precision and accuracy of measurements at the lowest
calibration standard; Figure 4b shows the chromatogram
derived from a derivatization reaction performed in sepa-
rate batches of drug-free rat brain homogenate, reporting
two peaks: Peak 1 represents DNPH (Rt = 3.43), whereas
Peak 2 represents HDZ (Rt = 5.64) formed by derivatiza-
tion of free MDA. Figure 5 hows chromatograms of homo-
genate brain samples of treated rats. In detail, Figure 5a
shows the measured concentration of HDZ (6.261 lg/ml)
corresponding to free MDA determined in rats treated with
PQ alone; Figure 5b shows the measured concentration of
Validation of analytical method
Calibration curves were obtained by plotting the peak area
of the unknown concentration versus that of the known
concentration of HDZ added to drug-free rat brain homo-
genate. The curves were constructed from four replicate
Figure 4 Representative chromatograms of homogenate rat brain sample (k = 310 nm): (a) sample of untreated rat; (b) derivatization reaction
performed in drug-free rat brain homogenate, reporting two peaks referred to DNPH and HDZ. Peak: 1 DNPH, Rt = 3.43; Peak 2 HDZ, Rt = 5.64.
6
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Milena Rizzo et al.
Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Figure 5 Representative chromatograms of homogenate brain sample of rats treated with PQ (150 lg/kg, icv) and polyphenol derivatives
10 lmol/kg, ip) (k = 310 nm), as follows: (a) rat treated with PQ alone; peak 1 DNPH Rt = 3.30; peak 2 HDZ Rt = 5.52; (b) rat treated with PQ
and Ole; peak 1 DNPH Rt = 3.38; peak 2 HDZ Rt = 5.48; (c) rat treated with PQ and AcHT; peak 1 DNPH Rt = 3.25; peak 2 HDZ Rt = 5.65.
(
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
7

Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Milena Rizzo et al.
measurements of nine concentrations (0.05-0.1-0.2-0.5-1-
2
standards by adding amounts of HDZ 0.1-0.2-0.5-1-2-4-6
and 8 lg/ml in replicate (n = 3). Recovery values ranged
from 94.1 to 105.8%, measured spiking 4 and 1 lg/ml,
respectively, as reported in Table 4.
-4-6-8 lg/ml). A linear response was observed over the
examined concentration range. The square of the correla-
tion coefficient of HDZ was 0.99953. Data of curve equa-
tion Y= A x + B are reported as follows: A = 80.074;
B = ꢁ2.380; standard error
error = 5.927. The method allowed the determination of
0 ng/ml of HDZ in brain homogenate samples with a sig-
V y = 4946; mean %
MDA determination
5
Table 5 shows the results of free and total MDA determina-
tion measured as HDZ in homogenate brain samples. Rats
were randomly divided into eight groups, each comprising
six animals, and were treated as follows: group 1-vehicle
(0.9% NaCl, icv); group 2-PQ alone (150 lg/kg, icv);
groups 3-8-PQ (150 lg/kg, icv) plus each of the six
nal-to-noise ratio of 10 (LOQ) and LOD of 5 ng/ml with a
signal-to-noise ratio of 3.
Precision and accuracy for within-day assay and
between-day assay were reported in Tables 2 and 3, respec-
tively. Data were determined on homogenate brain sam-
ples, derived from non-treated rats, by adding 0.1-0.2-0.5-
Table 4 Determination of extraction recovery of 2,4-dinitrophenyl-
hydrazone (HDZ) (0.1-0.2-0.5-1-2-4-6 and 8 lg/ml) from homogenate
brain samples derived from untreated rats (n = 3)
1
-2-4 and 6 lg/ml of HDZ in replicate (n = 3). In the
within-day assay, CV ranged from 0.48 to 8.33%, where the
relative mean error (RME) ranged from 0.02% to 0.11%. In
the between-day assay, the highest CV% was 9.95 and cal-
culated using 1 lg/ml of HDZ, where the highest RME%
was 0.16 and calculated using 0.1 lg/ml of HDZ.
Spiked
Recovered
concentration concentration
CV
RME
%
Recovery
%
HDZ lg/ml
HDZ lg/ml
SD
(%)
0
0
0
1
2
4
6
8
.1
.2
.5
0.095
0.189
0.526
1.058
1.935
3.762
6.072
7.644
0.001
0.004
0.003
0.088
0.110
0.116
1.05
0.11
0.09
95.0
94.3
Extraction efficiency was measured as recovery of HDZ
from aqueous standards and pooled brain homogenate
1.86
0.48
8.33
5.70
0.01 105.1
0.07 105.8
Table 2 Precision and accuracy for within-day assay of the studied
0.03
96.8
94.1
compound performed on homogenate brain samples by adding 0.1-
3.09 001
0
.2-0.5-1-2 and 4 lg/ml of 2,4-dinitrophenylhydrazone (HDZ) in repli-
0.203 334
0.159 2.09
0.01 101.2
cate (n = 3)
0.00
95.6
Spiked
Found
SD, Standard deviation; CV, Coefficient of variation; RME, Relative
mean error.
concentration
HDZ lg/ml
concentration
HDZ lg/ml
CV
RME
%
SD
(%)
0
0
0
1
2
4
.1
.2
.5
0.095
0.189
0.526
1.058
1.935
4.019
0.001 1.05
0.004 1.86
0.003 0.48
0.088 8.33
0.110 5.70
0.287 7.14
0.11
0.09
0.01
0.07
0.03
0.02
Table 5 Determination of free and total malondialdehyde (MDA)
measured as 2,4-dinitrophenylhydrazone (HDZ) in homogenate brain
samples obtained from rats divided into eight groups (n = 6) and trea-
ted with the tested compounds, as follows: group 1 with vehicle
(0.9% NaCl, icv); group 2 with paraquat (PQ) (150 lg/kg, icv); groups
3–8 with PQ plus polyphenol derivatives given intraperitoneally
(
10 lmol/kg), that is: oleuropein (Ole), acetylated oleuropein (AcOle),
SD, Standard Deviation; CV, Coefficient of variation; RME, Relative
mean error.
oleuropein aglycone as a mixture of tautomeric forms (OleAa-d),
acetylated oleuropein aglycone as a mixture of tautomeric forms
(
AcOleAa-d), hydroxytyrosol (HT), acetylated hydroxytyrosol (AcHT).
Table 3 Precision and accuracy for between-day assay of the stud-
Bound MDA is expressed in percentage
ied compound performed on homogenate brain samples by adding
Free MDA as
Total MDA as
HDZ
0
.1-0.2-0.5-1-2 and 4 lg/ml of 2,4-dinitrophenylhydrazone (HDZ) in
Group
of rats
Tested
HDZ
Bound
replicate (n = 3)
compounds lg/ml ꢀ SD
lg/ml ꢀ SD
MDA %
Spiked
Found
1
2
3
4
5
Vehicle
PQ
0.107 ꢀ 0.010 0.233 ꢀ 0.013 33
6.261 ꢀ 0.094 7.654 ꢀ 0.061 28
4.582 ꢀ 0.065 6.620 ꢀ 0.079 31
concentration
HDZ lg/ml
concentration
HDZ lg/ml
CV
RME
%
SD
(%)
Ole + PQ
0
0
0
1
2
4
.1
.2
.5
0.096
0.189
0.530
1.069
1.930
3.989
0.002 1.50
0.006 2.75
0.013 2.50
0.105 9.95
0.109 5.40
0.343 8.25
0.16
0.15
0.05
0.09
0.03
0.02
AcOle + PQ 4.219 ꢀ 0.047 6.226 ꢀ 0.071 32
OleAa-
4.127 ꢀ 0.044 6.027 ꢀ 0.088 32
d + PQ
6
AcOleAa-
d + PQ
2.413 ꢀ 0.025 3.814 ꢀ 0.041 32
7
8
HT + PQ
AcHT + PQ
1.913 ꢀ 0.021 2.509 ꢀ 0.028 24
1.796 ꢀ 0.010 2.362 ꢀ 0.063 24
SD, Standard Deviation; CV, Coefficient of variation; RME, Relative
mean error.
SD, standard deviation.
8
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Milena Rizzo et al.
Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
polyphenol derivatives, that is Ole, AcOle, OleAa-d, AcO-
leAa-d, HT and AcHT (10 lmol/kg, ip). A central inocula-
tion of herbicide PQ resulted in a strong enhancement of
MDA levels, in comparison with control rats. The intraperi-
toneal polyphenol administrations significantly decreased
free MDA levels (F = 1008E04; P < 0.001) and total MDA
levels (F = 1185E04; P < 0.001) in brain tissue, compared
to MDA levels determined in rats treated with PQ alone.
Bound MDA is expressed as a percentage. It was evident
that acetylated derivatives were more active than the
respective parent compounds (P < 0.001). Moreover,
acetylated hemiacetal aglycone product used as a mixture
of four tautomeric forms (AcOleAa-d) resulted in a reduc-
tion of MDA concentration. In particular, rats treated with
acetylated oleuropein aglycone showed a decrease in total
MDA of 70% versus a decrease in 24% for the correspond-
ing non-acetylated derivative.
assessing oxidative stress in-vitro and in-vivo model of dif-
[
26,27]
ferent diseases is considered to be best practice,
despite the differences between the analytical and validation
[
28,29]
methods.
Our fully validated study demonstrated the reliability of
MDA assessment using a HPLC associated with DAD.
Derivatization process was shown to be highly specific and
presented clear advantages. In our experience, hydrazone
derivative formation proceeds readily at room temperature
under mildly acidic pH conditions; moreover, the formed
HDZ is not unique to a given aldehyde, and therefore, a
solid-phase extraction procedure was established to improve
selectivity towards MDA-hydrazone derivative. In fact,
chromatographic separation of other DNPH derivatives as
short-chain water-soluble carbonylic compounds, namely
formaldehyde, acetaldehyde and acetone, was not seen.
Studying the stability of HDZ, we determined a signifi-
cant sensitivity to light; in fact, a reduction of up to 18% of
the initial MDA peak signal was observed within 1 h of
exposure to light. Subsequently, all of the samples were
protected from exposure to light during the derivatization
with DNPH and subsequent steps. Then, HDZ solutions
were stable for more than 12 h at room temperature.
The antioxidant activity of analysed polyphenols was as
follows: Ole<AcOle<OleAa-d<AcOleAa-d<HT<AcHT.
Discussion
The analytical method here described proved to be highly
sensitive and specific to determine the hydrazone derivative
obtained after the MDA derivatization process; it was fully
validated and provided good accuracy, precision and recov-
ery.
Because MDA may originate from different sources that
are mainly free and protein bound MDA, with our method
we could quantify total MDA using an alkaline hydrolysis
with 2 M NaOH (for 30 min under heating to 60 °C) which
was found to be optimal. Then, a determination of bound
MDA in rat brains was successfully ameliorated.
For the first time, we developed an original SPE extrac-
tion procedure applied to homogenate rat brain to deter-
mine HDZ as marker of induced oxidative stress.
To stimulate MDA formation in animal brains, an exper-
imental model described by Mollace et al. was
Different assays have been developed to evaluate the
antioxidant activity of plants and food constituents: The
use of an adequate assay is a critical point. There is a need
to agree governance on in-vitro antioxidant methods based
on an understanding of the mechanisms involved, because
some of the assays are done at non-physiological pH values.
In brief, the most widely used assay is MDA assay, which is
associated with lipid peroxidation. In biological matrices,
[
30]
employed.
They demonstrated that a microinfusion of
PQ into the Substantia Nigra produces brain damage via
the abnormal formation of oxygen free radicals.
Because the lipophilicity of a substance is one of the
parameters which influence its biological activity, natural
and semisynthetic derivatives of Olea europaea L. were
studied to describe their antioxidant properties and corre-
late their activity with lipophilicity. Then, a comparison of
experimental LogP values and theoretical partition coeffi-
cients calculated by two different procedures was per-
formed. All of the acetylated entities were reported to be
much more lipophilic than the corresponding starting spe-
cies. Moreover, we showed that all of the acetyl-derivatives
of phenolic compounds possessed an antioxidant activity
higher than their parent compounds. In particular, a mix-
ture of tautomeric forms of acetylated Ole aglycone had an
enhanced capacity to antagonize MDA formation, com-
pared to deacetylated derivative. AcHT had more effective
antioxidant activity than deacetylated form HT. The results
obtained using aglycone forms suggest the presence of an
active substance that can be naturally produced in vivo by
enzymatic Ole hydrolysis. Otherwise, Ole showed only a
MDA exists both free and bound to -SH and -NH groups
2
of macromolecules, that is, proteins and nucleic acids and
only low amounts of free MDA were detected in biological
[
23]
samples.
its reaction with thiobarbituric acid (TBA) in spectropho-
In the past, MDA was mostly determined after
[
24]
tometry and measuring absorbance at 532 nm.
Although it has been considered a favourable assay because
of its ease of use, it is lacking in specificity. TBA reacts not
only with MDA but with many other compounds of biolog-
ical origin; identical MDA-TBA adducts were yielded from
non-lipid-related materials. An important review identified
gas and liquid chromatography-based analyses as the most
widely used techniques for MDA determination in different
matrices, as well as several derivatization-based strate-
[
25]
gies.
Even now, the use of MDA as a biomarker for
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
9

Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
Milena Rizzo et al.
reduced protective action towards PQ-induced oxidative
stress.
Conclusion
As shown in Table 5, a small amount of MDA was
detected in the control rats treated only with vehicle. The
concentrations 0.157 and 0.233 lg/ml of HDZ of free and
total MDA, respectively, were considered to be physiologi-
cal and were significantly different to the PQ-treated levels
This study was performed to assess how the neurotoxic
effect induced by central infusion of PQ in rats is antago-
nized by a pretreatment with antioxidant compounds
derived from Olea europaea L., given intraperitoneally as
natural and semisynthetic polyphenols with different
lipophilicity. The lipophilic character of the starting species
was significantly improved by their acetylation, as proved
by Log P calculations.
(
6.261 and 8.654 lg/ml of HDZ of free and total MDA,
respectively). Both natural and semisynthetic derivatives of
Olea europaea L. showed to possess an antioxidant activity.
HT was able to reduce both free MDA (1.913 lg/ml)
and total MDA (2.509 lg/ml). This result could be related
to catecholic moiety. It acts by breaking the radical chain
Pretreatment with oleuropein, oleuropein aglycone,
hydroxytyrosol and related acetyl-derivatives, able to cross
the brain–blood barrier even given systemically, antago-
nized an exaggerated formation of reactive oxygen species.
Most importantly, all of the peracetylated derivatives
exerted a higher antioxidant activity than the parent com-
pounds.
and forming
a phenoxyl radical, stabilized by an
intramolecular hydrogen bond with the free hydrogen of
the ortho-hydroxyl group. Nevertheless, the antioxidant
properties of o-diphenolic moiety seem to be insufficient to
ensure adequate protection against PQ-induced stress,
pointing to the decisive role played by the aglycone tau-
tomeric mixture’s dialdehydic function.
The results confirm the hypothesis that a major lipophi-
lic character, improving biological availability, can help to
ensure enhanced activity of the molecules.
Moreover, a lowest concentration of free MDA (1.796 lg/
ml) and total MDA (2.362 lg/ml) was determined in rat
treated with AcHT. Interestingly, the reported data confirm
the hypothesis that a major lipophilic character can help to
ensure improved biological availability of the molecules.
Indeed, all the acetylated compounds (AcOle, AcOleAa-d
and AcHT) exerted a better protective action than their
hydrophilic analogues. These species are supposed to better
cross the blood–brain barrier and biochemically be con-
verted in vivo into their original active forms.
Declaration
All animal experiments were carried out in accordance with
the EU Directive 2010/63/EU for animal.experiments.
(Http://ec.europa.eu/environment/chemicals/lab_animals/
legislation_en.Htm). All experimental protocols were car-
ried out the University of Magna Graecia’s (Catanzaro,
Italy) animal testing facility after approval by the Local
Ethics Committee.
vitamin E-enriched beverage dietary 10. Romero-Garc ꢁı a JM et al. Obtaining
References
supplementation on inflammation
associated to exercise and age. Nutri-
ents 2016; 8: 619.
sugars and natural antioxidants from
olive leaves by steam-explosion. Food
Chem 2016; 210: 457–465.
1. Grosso G et al. A comprehensive
meta-analysis on evidence of Mediter-
ranean diet and cardiovascular disease:
are individual components equal?, Crit
Rev Food Sci Nutr 2017; 57: 3218–
6. Ghanbari R et al. Valuable nutrients and 11. Carrasco-Pancorbo L. et al. Evaluation
functional bioactive in different parts of
olive (Olea europaea L.)-a review. Int J
Mol Sci 2012; 13: 3291–3340.
of the antioxidant capacity of individ-
ual phenolic compounds in virgin
olive oil. J Agric Food Chem 2005; 53:
8918–8925.
3232.
2
. Valls-Pedret C et al. Mediterranean
diet and age-related cognitive decline:
a Randomized clinical trial. JAMA
Intern Med 2015; 175: 1094–1103.
7. Khalatbary AR. Olive oil phenols and
neuroprotection (Review). Nutr Neu- 12. Casamenti F. Oleuropein Aglycone: a
rosci 2013; 16: 243–249.
possible drug against degenerative condi-
tions In vivo evidence of its effectiveness
against Alzheimer Disease (Review). J
Alzheimers Dis 2015; 45: 679–688.
8. Lee O, Lee B. Antioxidant and
antimicrobial activities of individual
and combined phenolics in Olea euro-
3
. Covas MI et al. Virgin olive oil: a key
food for cardiovascular risk protection.
Br J Nutr 2015; 113(Suppl. 2):S19–S28.
. Cicerale S et al. Biological activities of
phenolic compounds present in virgin
olive oil. Int J Mol Sci 2010; 11: 458–
paea leaf extract. Bioresour Technol 13. Fitoꢁ
M
et al. Bioavailability and
4
2010; 101: 3751–3754.
antioxidant effects of olive oil pheno-
lic compounds in humans: a review.
Ann Ist Super Sanit ꢀa 2007; 43: 375–
381.
9. Vogel P et al. VT. Zani, D. de Souza,
S. Morelo Dal Bosco, Polyphenols
benefits of olive leaf (Olea europaea
479.
5
. Cap oꢁ X et al. Effects of almond and
L) to human health (Review). Nutr 14. Serra L et al. Maci ꢀa , MP. Romero,
Hosp 2014; 31: 1427–1433. MJ. Motilva, Distribution of olive oil
olive oil-based docosahexaenoic- and
1
0
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Milena Rizzo et al.
Antioxidant activity of oleuropein and semisynthetic acetyl-derivatives
phenolic compounds in rat tissues 19. Bulotta S et al. Antiproliferative and 26. Nistic oꢀ S et al. Effect of MN (III) tetradi
after administration of
a
phenolic
antioxidant effects on breast cancer
cells of Oleuropein and its semisyn-
thetic peracetylated derivatives. Food
Chem 2011; 127: 1609–1614.
(4-benzoic acid) porphyrin by photody-
namically generated free radicals on
SODs keratinocytes. J Biol Regul Homeost
Agents 2013; 27: 781–790.
extract from olive cake. Mol Nutr
Food Res 2012; 56: 486–496.
5. Procopio G et al. Chemical-catalytic
1
1
method for the peracylation of Oleu- 20. Capasso R et al. A highly convenient 27. Muscoli F et al. Olea Europea-derived
ropein and its products of hydrolysis.
International Patent PCT/IT2008/
synthesis of hydroxytyrosol and its
recovery from agricultural waste waters.
J Agric Food Chem 1999; 47: 1745–1748.
phenolic products attenuate antinoci-
ceptive morphine tolerance: an inno-
vative strategic approach to treat
cancer pain. J Biol Regul Homeost
Agents 2014; 28: 809–820.
000303.
6. Procopio S et al. Synthesis, biological 21. www.actelion.com/uninet.
evaluation and molecular modelling 22. www.molinspiration.com/cgi_bin/prop
of oleuropein and its semisynthetic
erties.
28. Singh Z et al. Use of malondialdehyde
as a biomarker for assessing oxidative
stress in different disease pathologies:
a review. Iran J Public Health 2014; 43
(Suppl. 3): 7–16.
derivatives as cyclooxygenase inhibi- 23. Pilz J et al. Measurement of free and
tors. J Agric Food Chem 2009; 57:
1161–11167.
7. Impellizzeri D et al. The effects of a
bound malondialdehyde in plasma by
high performance liquid chromatogra-
phy as the 2,4-dinitrophenylhydrazine.
J Chromatogr B 2000; 742: 315–325.
1
1
polyphenol present in olive oil, Oleu-
29. Khoubnasab Jafari M et al. Comment
on “Use of malondialdehyde as a bio-
marker for assessing oxidative stress
ropein aglycone, in an experimental 24. Carbonneau MA et al. Free and
model of spinal cord injury in mice.
bound malondialdehyde measured as
thiobarbituric acid adduct by HPLC
in serum and plasma. Clin Chem
1991; 37: 1423–1429.
Biochem Pharmacol 2012; 83: 1413–
in different disease pathologies: a
1426.
review”. Iran J Public Health 2015; 44:
714–715.
1
8. Impellizzeri D et al. Oleuropein agly-
cone, an olive oil compound, amelio- 25. Giera M et al. Recent advancements 30. Mollace V et al. The role of oxidative
rates development of arthritis caused
by injection of collagen type II in
mice. J Pharmacol Exp Ther 2011; 339:
in the LC- and GC-based analysis of
malondialdehyde (MDA), a brief over-
view. Chromatographia 2012; 75: 433–
440.
stress in Paraquat-induced neurotoxic-
ity in rats: protection by non peptidyl
superoxide dismutase mimetic. Neu-
rosci Lett 2003; 335: 163–166.
859–869.
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
11