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Recently, a number of studies on health benefits associated with natural compounds have been demonstrated. Phenolics in fruits, vegetables, herbs and spices possess potent antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic activities. The fringe tree (Chionanthus virginicus) is used as a raw material by pharmaceutical industries for the preparation of homeopathy tinctures. The potential antioxidant activities of two secoiridoids from root bark of fringe tree (Chionanthus virginicus L.) were investigated to evaluate their potential value as the natural products for foods or cosmetic applications. In this study, antioxidant activities were measured by 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) scavenging, superoxide anion (O 2·−) radical scavenging, total antioxidant activity, reducing activity, hydrogen peroxide (H2O2) scavenging and ferrous metal chelating activity assays. These secoiridoids, as antioxidants neutralized the activities of radicals and inhibited the peroxidation reactions of linoleic acid emulsion. Total antioxidant activity was measured according to ferric thiocyanate method. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox, a water-soluble analog of tocopherol, were used as the reference antioxidant compounds. Ligustroside (3.70 × 10−3 M) and oleuropein (3.80 × 10−3 M) showed 71.9, 82.4, 80.7 and 90.4% inhibition on lipid peroxidation of linoleic acid emulsion, at the concentrations of 10 and 20 μg/mL. On the other hand, 20 μg/mL of standard antioxidant such as α-tocopherol (4.64 × 10−3 M), trolox (7.98 × 10−3 M), BHA (10.08 × 10−3 M) and BHT (9.06 × 10−3 M) exhibited 61.5, 29.8, 74.4 and 71.2% inhibition on peroxidation of linoleic acid emulsion, respectively. In addition, ligustroside and oleuropein had effective DPPH·, ABTS·+ and superoxide anion radicals scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities. Also, those various antioxidant activities were compared to BHA and BHT, α-tocopherol and trolox that are references antioxidants.
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ORIGINAL
Antioxidant secoiridoids from fringe tree
(Chionanthus virginicus L.)
_
Ilhami Gu
¨lc¸in ÆRiad Elias ÆAkc¸ahan Gepdiremen Æ
Khalil Taoubi ÆEkrem Ko
¨ksal
Received: 29 November 2007
ÓSpringer-Verlag 2008
Abstract Recently, a number of studies on health benefits associated with natural
compounds have been demonstrated. Phenolics in fruits, vegetables, herbs and
spices possess potent antioxidant, anti-inflammatory, antimutagenic and anticarci-
nogenic activities. The fringe tree (Chionanthus virginicus) is used as a raw material
by pharmaceutical industries for the preparation of homeopathy tinctures. The
potential antioxidant activities of two secoiridoids from root bark of fringe tree
(Chionanthus virginicus L.) were investigated to evaluate their potential value as the
natural products for foods or cosmetic applications. In this study, antioxidant
activities were measured by 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH)
scavenging, superoxide anion (O
2
-
) radical scavenging, total antioxidant activity,
reducing activity, hydrogen peroxide (H
2
O
2
) scavenging and ferrous metal chelating
activity assays. These secoiridoids, as antioxidants neutralized the activities of
radicals and inhibited the peroxidation reactions of linoleic acid emulsion. Total
antioxidant activity was measured according to ferric thiocyanate method. Butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), a-tocopherol and trolox, a
water-soluble analog of tocopherol, were used as the reference antioxidant com-
pounds. Ligustroside (3.70 910
-3
M) and oleuropein (3.80 910
-3
M) showed
71.9, 82.4, 80.7 and 90.4% inhibition on lipid peroxidation of linoleic acid
_
I. Gu
¨lc¸in (&)E. Ko
¨ksal
Department of Chemistry, Faculty of Science and Arts,
Atatu
¨rk University, 25240, Erzurum, Turkey
e-mail: igulcin@atauni.edu.tr; igulcin@yahoo.com
R. Elias K. Taoubi
Laboratory of Pharmacognosy and Homeopathy,
Pharmacy Faculty of Mediterranean University, 13385 Marseille, France
A. Gepdiremen
Department of Pharmacology, Medical Faculty, Abant _
Izzet Baysal University,
14280 Go
¨lko
¨y, Bolu, Turkey
123
Wood Sci Technol
DOI 10.1007/s00226-008-0234-1
emulsion, at the concentrations of 10 and 20 lg/mL. On the other hand, 20 lg/mL
of standard antioxidant such as a-tocopherol (4.64 910
-3
M), trolox
(7.98 910
-3
M), BHA (10.08 910
-3
M) and BHT (9.06 910
-3
M) exhibited
61.5, 29.8, 74.4 and 71.2% inhibition on peroxidation of linoleic acid emulsion,
respectively. In addition, ligustroside and oleuropein had effective DPPH, ABTS
?
and superoxide anion radicals scavenging, hydrogen peroxide scavenging, total
reducing power and metal chelating on ferrous ions activities. Also, those various
antioxidant activities were compared to BHA and BHT, a-tocopherol and trolox that
are references antioxidants.
Introduction
Oxidation processes are very important for the living organism. Uncontrolled
production of reactive oxygen species (ROS) and the unbalanced mechanism of
antioxidant protection result in onset of many diseases and accelerate aging. ROS
are a class of highly reactive molecules formed during aerobic life in living
organisms and include superoxide anion radicals (O
2
-
), hydroxyl radicals (OH)
and non free-radical species such as H
2
O
2
and singlet oxygen (
1
O
2
) (Halliwell and
Gutteridge 1989;Gu
¨lc¸in et al. 2002a,b). There is a balance between the generation
of ROS and inactivation of ROS by the antioxidant systems in organisms. ROS
leads to oxidative modification in cellular membrane or intracellular molecules if
there is no balance between ROS and antioxidant defence mechanisms (Duh et al.
1999;Bu
¨yu
¨kokurog
˘lu et al. 2001;Gu
¨lc¸in et al. 2003a). In addition, under
pathological conditions or oxidative stress, ROS are overproduced and result in
peroxidation of membrane lipids, leading to the accumulation of lipid peroxides.
However, they are removed by antioxidant defence mechanisms. Antioxidants are
considered as possible protection agents to reduce oxidative damage of human body
from ROS and retard the progress of many chronic diseases as well as lipid
peroxidation (Pryor 1991; Kinsella et al. 1993; Lai et al. 2001;Gu
¨lc¸in et al. 2003a).
Therefore, there is a growing interest in the substances exhibiting antioxidant
properties that are supplied to human and animal organisms as food components or
as specific pharmaceutics. Antioxidants may be defined as compounds that inhibit or
delay the oxidation of other molecules by inhibiting the initiation or propagation of
oxidizing chain reactions (Velioglu et al. 1998;Gu
¨lc¸in et al. 2006a).
BHA, BHT, propylgallate and tert-butylhydroquinone are the most commonly
used antioxidants. However, their safety is questioned due to their toxicity and
possible carcinogenicity, liver damage and carcinogenesis (Wichi 1988; Sherwin
1990; Sun and Fukuhara 1997;Gu
¨lc¸in et al. 2003a). Hence, in the recent years, the
restriction in the use of synthetic antioxidants, such as BHA and BHT, has caused an
increased interest towards natural antioxidant substances (Baardseth 1989; Oktay
et al. 2003;Gu
¨lc¸in et al. 2005). Therefore, much attention has been focused on
natural antioxidants. These antioxidants occur in all higher plants and in all parts of
the plant, such as wood, bark, stems, pods, leaves, fruits, roots, flowers and seeds
(Al-Ismail and Aburjai 1989). Thus, development of safer natural antioxidants that
can replace synthetic ones has been of interest (Liyana-Pathirana and Shahidi 2006).
Wood Sci Technol
123
Chionanthus virginicus L., fringe tree, is a shrub of the eastern America. This
Oleaceae is used in folk medicine as cholagogue, diuretic and tonic (Duke and Wain
1981). The leaves contain flavonoids, such as rutin, kaempferol-3-glucoside,
kaempferol-3-rutinoside, quercetin triglycosides (Harborne and Green 1980) and
triterpenoid compounds such as ursolic acid (Pourra et al. 1954). Stem and root
barks contain a lignin, phillyrin (Steinegger and Jacober 1959). Nowadays, root bark
is used in homeopathy for hepatitis (Guermonprez et al. 1997).
Oleuropein, and ligustroside are the most abundant secoiridoid glucosides.
Oleuropein, a non-toxic secoiridoid, is a powerful antioxidant and antiangiogenic
agent with anticancer effect (Hamdi et al. 2003) that actively scavenges reactive
oxygen (Manna et al. 2002) and nitrogen species (De la Puerta et al. 2001), inducing
the production of nitric oxide in macrophages (Visioli et al. 1998). Oleuropein had a
potent antitumor agent with direct effects against tumor cells (Hamdi and Castellon
2005). Two secoiridoid glucosides, ligustroside and oleuropein were isolated from
the fruits of Ligustrum lucidum and their structures were elucidated by spectro-
scopic methods (He et al. 2001).
As mentioned above ligustroside and oleuropein have been isolated from
different plant species, but no information has been found about in vitro antioxidant
and antiradical activities of ligustroside and oleuropein from root bark of fringe tree
(Chionanthus virginicus L.). The main objectives of the present study were to assess
the antioxidant potential of ligustroside and oleuropein from root bark of fringe tree
(Chionanthus virginicus L.) in different in vitro antioxidant assays including total
antioxidant activity determination, DPPH free radical scavenging, ABTS radical
scavenging, reducing power, superoxide anion radical scavenging, hydrogen
peroxide scavenging and metal chelating activities.
Materials and methods
Chemicals
2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), nicotinamide adenine
dinucleotide (NADH), butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), the stable free
radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH), linoleic acid, 3-(2-Pyridyl)-5,6-bis
(4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), 6-hydroxy-2,5,7,8-tetrame-
thylchroman-2-carboxylic acid (Trolox), ethylenediaminetetraacetic acid (EDTA),
a-tocopherol, polyoxyethylenesorbitan monolaurate (Tween-20), and trichloroacetic
acid (TCA) were obtained from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany).
Ammonium thiocyanate was purchased from Merck. All other chemicals used were of
analytical grade and were obtained from either Sigma-Aldrich or Merck.
Plant materials
Samples of dried root bark of Chionanthus virginicus were provided by BOIRON
laboratories (No: 97070214, 97070425 and 97030136). They were collected in
Wood Sci Technol
123
south-east of USA and dried naturally on site. Root bark was powdered and stored in
a dry place and protected from light until used.
Extraction, isolation and identification of secoiridoids
Powdered root bark of Chionanthus virginicus (100 g) were extracted with 300 ml
of MeOH twice under reflux during 30 min. The extract was concentrated in rotary
evaporator. The residue was dissolved in 300 mL of distilled water and was
successively extracted with 4 9100 mL of AcOEt. Organic phase was evaporated
in rotary evaporator to give residues of 4.4 g. This residue was subjected to LPLC
(low pressure liquid chromatography) on Chromatospac Prwep 10 (Jobin Yvon)
with a 40 9500 mm column filled with Lichroprep RP-18 (25–40 lm, 200 g,
Merck). The gradient solvent system was MeOH/H
2
O (v/v): 30/70 (700 mL); 40/60
(500 mL); 50/50 (500 mL); 60/40 (500 mL); 70/30 (500 mL); 80/20 (500 mL); 90/
10 (500 mL); 100/0 (1000 mL). Collected fractions (100 mL) were examined by
TLC; it was performed on silica gel 60F254 (Merck) with EtOAc/HCOOH/HOAc/
H
2
O (100:11:11:27). Plates were examined under UV light at 254 and 366 nm; then
they sprayed with H
2
SO
4
solution (20% MeOH).
Two main compounds, oleuropein (MeOH 45%; 1230.9 mg) and ligustroside
(MeOH 50%; 297.5 mg) were isolated from AcOEt extract. Chemical structures of
both the secoiridoids are shown in Fig. 1. Structure elucidation was carried out
using spectroscopic methods: LC-UV, LC-MS (mass spectrophotometer with
negative ion detection),
1
H NMR (DRX Bru
¨ker 500 spectrophotometer operating at
500 MHz), and
13
C NMR (DRX Bru
¨ker 500 spectrophotometer operating at
125 MHz). NMR measurements were performed in CD
3
OD or in DMSO-d6.
HPLC-MS, HPLC analysis and identification of secoiridoids compounds have been
described in previous report (Boyer et al. 2005).
Total antioxidant activity: ferric thiocyanate method
The antioxidant activity of ligustroside, oleuropein and standards was determined
according to the ferric thiocyanate method in linoleic acid emulsion (Mitsuda et al.
1996). A stock solution contained 10 mg of ligustroside and oleuropein dissolved in
10 mL deionized water. Different concentrations of stock ligustroside, and
oleuropein solution samples (10–20 lg/mL) were prepared by diluting the stock
solution in 2.5 mL of potassium phosphate buffer (0.04 M, pH 7.0) and these were
added to 2.5 mL of linoleic acid emulsion in potassium phosphate buffer (0.04 M,
pH 7.0). The mixed solution (5 mL) was incubated at 37°C in glass flask. At regular
intervals during incubation, 0.1 ml aliquot of the mixture was diluted with 3.7 ml of
ethanol, followed by the addition of 0.1 ml of 30% ammonium thiocyanate and
0.1 ml of 20 mM ferrous chloride in 3.5% hydrochloric acid. The peroxide level
was determined by reading the absorbance at 500 nm in a spectrophotometer
(CHEBIOS s.r.l. UV-VIS). During linoleic acid oxidation, peroxides that oxidize
Fe
2?
to Fe
3?
are formed. The latter ions form a complex with thiocyanate and this
complex has a maximum absorbance at 500 nm. These steps were repeated every
12 h until the control reached its maximum absorbance value. Therefore, high
Wood Sci Technol
123
absorbance indicates high linoleic acid emulsion oxidation. Solutions without added
samples were used as blanks. All data on total antioxidant activity are the average of
duplicate experiments. The percentage of inhibition of lipid peroxidation in linoleic
acid emulsion was calculated by following equation:
Inhibition of lipid peroxidation (%) = 100 AS
AC100

where A
c
is the absorbance of the control reaction and A
s
is the absorbance in the
presence of the sample of ligustroside, oleuropein or other test compounds. In the
control, the sample was replaced with an equal volume of ethanol (Gu
¨lc¸in et al.
2004a;Gu
¨lc¸in 2006a).
Total reduction activity by Fe
3?
–Fe
2?
transformation
The samples prepared for ferric thiocyanate method was used for the present and
other antioxidant assays. The reducing activities of ligustroside and oleuropein were
determined by the method of Oyaizu (1986). The capacity of ligustroside and
oleuropein to reduce the ferric–ferricyanide complex to the ferrous–ferricyanide
complex of Prussian blue was determined by recording the absorbance at 700 nm
O
OOO
OH
O
O
OH
OH
OH
OH
Ligustroside
O
OOO
OH
O
O
OH
OH
OH
OH
Oleurop ein
OH
Alpha-tocopherol Trolox
O
HO
OH
O
O
HO
OCH 3
OH OH
CH3
BHA BHT
Fig. 1 Chemical structures of ligustroside and oleuropein from root bark of fringe tree (Chionanthus
virginicus L.) and standard antioxidants like BHA,BHT,a-tocopherol and trolox (BHA butylated
hydroxyanisole, BHT butylated hydroxytoluene)
Wood Sci Technol
123
after incubation. Simply, different concentrations of ligustroside and oleuropein
(10–20 lg/mL) in 1 mL of distilled water were mixed with phosphate buffer
(2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K
3
Fe(CN)
6
] (2.5 mL, 1%).
The mixture was incubated at 50°C for 20 min. Aliquots (2.5 mL) of trichloroacetic
acid (10%) were added to the mixture, which was then centrifuged for 10 min at
10009g(MSE Mistral 2000, UK). The upper layer of solution (2.5 mL) was mixed
with distilled water (2.5 mL) and FeCl
3
(0.5 mL, 0.1%), and the absorbance was
measured at 700 nm in a spectrophotometer. In the control, the sample was replaced
with an equal volume of ethanol. Increased absorbance of the reaction mixture
indicates grater reduction capability (Elmastas¸ et al. 2006a).
Metal chelating activity on ferrous ion
Ferrous ion (Fe
2?
) chelation by ligustroside, oleuropein and standards was
estimated by the Ferrozine assay (Dinis et al. 1994). Briefly, 0.4 mL of ligustroside
and oleuropein (10 lg/mL) was added to a solution of 2 mM FeCl
2
(0.05 mL). The
reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the total
volume was adjusted to 4 mL with ethanol. Then, the mixture was shaken
vigorously and left for waiting at room temperature for 10 min. After the mixture
had reached the equilibrium, the absorbance of the solution was then measured
spectrophotometrically at 562 nm. The results were expressed as percentage of
inhibition of the ferrozine–Fe
2?
complex formation. The percentage of inhibition of
ferrozine–Fe
2?
complex formation was calculated using the formula given below:
Ferrous ion chelating effect (%) = ACAS
AC

100
where A
C
is the absorbance of the ferrozine–Fe
2?
complex and A
S
is the absorbance
in the presence of the sample of ligustroside and oleuropein or standards (Gu
¨lc¸in
et al. 2004b; Elmastas et al. 2006b).
Hydrogen peroxide scavenging activity
The hydrogen peroxide scavenging ability of ligustroside and oleuropein was
determined according to the method of Ruch et al. (1989). A solution of H
2
O
2
(40 mM) was prepared in phosphate buffer (pH 7.4). Ligustroside and oleuropein at
the 10 lg/mL concentration in 3.4 mL phosphate buffer was added to a H
2
O
2
solution (0.6 mL, 40 mM). The absorbance value of the reaction mixture was
recorded at 230 nm. A blank solution contained the phosphate buffer without H
2
O
2
.
The percentages of H
2
O
2
scavenging of ligustroside, oleuropein and standard
compounds were calculated as:
H2O2scavenging effect (%) = ACAS
AC

100
Wood Sci Technol
123
where A
C
is the absorbance of the control, and A
S
is the absorbance in the presence
of the sample of ligustroside and oleuropein or standards (Elmastas et al. 2006c;
Gu
¨lc¸in et al. 2006a).
ABTS radical cation decolorization assay
The spectrophotometric analysis of ABTS
?
radical scavenging activity was
determined according to Re et al. (1999). The ABTS
?
cation radical was produced
by the reaction between 7 mM ABTS in H
2
O and 2.45 mM potassium persulfate,
stored in the dark at room temperature for 12 h. Before usage, the ABTS
?
solution
was diluted to get an absorbance of 0.700 ±0.025 at 734 nm with phosphate buffer
(0.1 M, pH 7.4). As mentioned above, the ABTS
?
was generated by incubating
ABTS with potassium persulfate. Chemical compounds that inhibit the potassium
persulfate activity may reduce the production of ABTS
?
. This reduction results in a
decrease of the total ABTS
?
in the system and contributes to the total ABTS
?
scavenging capacity.
For stock solutions of 10 mg of ligustroside and oleuropein was dissolved in
10 ml distilled water. Then, 1 ml of ABTS
?
solution was added to 3 mL of
ligustroside and oleuropein solution in ethanol at different concentrations (10–
20 lg/mL). Thirty minutes later, the percentage of inhibition in the absorbance at
734 nm was calculated for each concentration relative to a blank absorbance
(ethanol). All determinations were carried out at least three times, and in triplicate.
The ABTS
?
concentration (mM) in the reaction medium was calculated from the
following calibration curve, determined by linear regression (R
2
: 0.9922):
Absorbance ¼0:0116 [ABTSþþ0:0479:
The capability to scavenge the ABTS
?
radical was calculated using the
following equation:
ABTSþ scavenging effect (%) = ACAS
AC

100
where A
C
is the initial concentration of the ABTS
?
and A
S
is absorbance of the
remaining concentration of ABTS
?
in the presence of scavengers (Gu
¨lc¸in et al.
2006b;Gu
¨lc¸in 2006b).
1, 1-Diphenyl-2-picryl-hydrazil free radical scavenging activity
1, 1-Diphenyl-2-picryl-hydrazil (DPPH) free radical scavenging activities for
ligustroside and oleuropein were measured from the bleaching of purple colored
methanol solution of the stable DPPH radical, following the method described by
Blois (1958). The capacity of ligustroside and oleuropein to scavenge the lipid-
soluble DPPH radical, which results in the bleaching of the purple color exhibited
by the stable DPPH radical, is monitored at an absorbance of 517 nm. Basically,
0.1 mM ethanolic solution of DPPHwas prepared daily. Then, 1 ml of this solution
was added to 3 mL of ligustroside and oleuropein solution in ethanol at different
Wood Sci Technol
123
concentrations (10–20 lg/mL). Thirty minutes later, the absorbance was measured
at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical
scavenging activity. The DPPHconcentration (mM) in the reaction medium was
calculated from the following calibration curve, determined by linear regression
(R
2
: 0.9974):
Absorbance ¼5:869 104DPPH½þ0:0134:
The capability to scavenge the DPPHradical was calculated using the following
equation:
DPPH scavenging effect (%) = ACAS
AC

100
where A
C
is the initial concentration of the stable DPPH radical without the test
compound and A
S
is absorbance of the remaining concentration of DPPHin the
presence of ligustroside and oleuropein (Gu
¨lc¸in et al. 2004c,2007; Cristiane de
Souza et al. 2004).
Superoxide anion radical scavenging activity in PMS-NADH/NBT systems
Measurement of superoxide anion scavenging activity of ligustroside and oleurop-
ein was based on the method described by Liu et al. (1991). Superoxide radicals are
generated in PMS-NADH systems by oxidation of NADH and assayed by the
reduction of NBT. In these experiments, the superoxide radicals were generated in
3 mL of Tris–HCl buffer (16 mM, pH 8.0) containing 1 mL of NBT (50 lM)
solution, 1 mL NADH (78 lM) solution and sample solution of ligustroside and
oleuropein (30 lg/mL) in water. The reaction was started by adding 1 mL of PMS
solution (10 lM) to the mixture. The reaction mixture was incubated at 25°C for
5 min and the absorbance was measured against blank samples at 560 nm. L-
Ascorbic acid was used as a positive control. Decreased absorbance of the reaction
mixture indicates increased superoxide anion radical scavenging activity. The
percentage inhibition of superoxide anion generation was calculated using the
following formula:
Superoxide scavenging effect (%) = ACAS
AC

100
where A
C
is the absorbance of the L-ascorbic acid, and A
S
is the absorbance of
ligustroside and oleuropein or standards (Gu
¨lc¸in et al. 2004d;Gu
¨lc¸in and Das¸ tan
2007).
Statistical analysis
All the analyses on total antioxidant activity were done in duplicate sets. The other
analyses were performed in triplicate. The data were recorded as mean ±standard
deviation and analyzed by SPSS (version 11.5 for Windows 98, SPSS Inc.). One-
way analysis of variance was performed by ANOVA procedures. Significant
Wood Sci Technol
123
differences between means were determined by LSD tests. Pvalues \0.05 were
regarded as significant and pvalues \0.01 very significant.
Results and discussion
Natural antioxidants have biofunctionalities such as the reduction of chronic
diseases like DNA damage, mutagenesis, carcinogenesis, etc., and inhibitions of
pathogenic bacteria growth, which are often associated with the termination of free
radical propagation in biological systems (Zhu et al. 2002). Thus, for medicinal
bioactive components, antioxidant capacity is widely used as a parameter. A number
of assays have been introduced to measure the total antioxidant activity of pure
compounds (Miller et al. 1996).
In this study, the antioxidant activity of the ligustroside and oleuropein were
compared to BHA, BHT, a-tocopherol and its water-soluble analog trolox. The
antioxidant activity of the ligustroside, oleuropein, a-tocopherol, trolox, BHA and
BHT was also evaluated in a series of the following in vitro tests: DPPH free
radical, ABTS radical and superoxide anion radicals scavenging, total antioxidant
activity by ferric thiocyanate method, reducing activity, hydrogen peroxide
scavenging activity and metal chelating activity.
Total antioxidant activity determination in linoleic acid emulsion system by
ferric thiocyanate method
The effects of various concentrations of ligustroside and oleuropein (from 10 to
20 lg/mL) on lipid peroxidation of linoleic acid emulsion are shown in Fig. 2and
were found to be 71.9 and 82.4 (for ligustroside), 80.7 and 90.4% (for oleuropein),
0
1
2
3
0 122436486
Incubation time (Hour)
Lipid peroxidation (500 nm)
0
Control
α-Tocopherol-20 µg/mL
Trolox-20 µg/mL
BHA-20 µg/mL
BHT-20 µg/mL
Ligustroside-10 µg/mL
Ligustroside-20 µg/mL
Oleuropein-10 µg/mL
Oleuropein-20 µg/mL
Fig. 2 Total antioxidant activity of different concentrations (10–20 lg/mL) of ligustroside and
oleuropein from root bark of fringe tree (Chionanthus virginicus L.), a-tocopherol and trolox (20 lg/mL)
Wood Sci Technol
123
respectively. On the other hand, a-tocopherol (4.64 910
-3
M), trolox
(7.98 910
-3
M), BHA (10.08 910
-3
M) and BHT (9.06 910
-3
M) exhibited
61.5, 29.8, 74.4 and 71.2% inhibition on peroxidation of linoleic acid emulsion,
respectively, at the 20 lg/mL concentration. The results clearly showed that
ligustroside and oleuropein had higher total antioxidant activity than a-tocopherol,
trolox, BHA and BHT at the same concentration (20 lg/mL). In the present study,
we determined the antioxidant activity of the ligustroside, and oleuropein in a
concentration-dependent manner. In a recent study, the antioxidant activity of
different lignans was evaluated in a time, temperature and concentration-dependent
manner. Also, antioxidant activity of the lignans had been investigated in a
structure–activity relationship study (Eklund et al. 2005).
Total reductive capability using the potassium ferricyanide reduction method
Figure 3depicts the reducing activity of the ligustroside, oleuropein and standards
(BHA, BHT, a-tocopherol and trolox) using the potassium ferricyanide reduction
method. For the measurements of the reductive activity, the Fe
3?
–Fe
2?
transfor-
mation was investigated in the presence of ligustroside and oleuropein using the
method of Oyaizu (1986). The reducing activity of ligustroside, oleuropein, a-
tocopherol and trolox increased with increasing concentration of samples. As can be
seen in the Fig. 3, ligustroside and oleuropein showed more effective reducing
activity than control at different concentrations (R
2
=9962, R
2
=9950). These
differences were statistically significant (p\0.01). Reducing power of ligustroside,
oleuropein and standard compounds are as follows: BHA [BHT [oleurop-
ein [a-tocopherol [ligustroside [trolox.
0
0.5
1
1.5
2
2.5
100
Concentration (
µg
/mL)
Absorbance (700 nm)
20
α-Tocopherol
Trolox
BHA
BHT
Ligus troside
Oleuropein
Fig. 3 Total reductive potential of different concentrations (10–20 lg/mL) of ligustroside, oleuropein
from root bark of fringe tree (Chionanthus virginicus L.), a-tocopherol and trolox
Wood Sci Technol
123
Ferrous ions chelating capacity
Transition metals have a major role in the generation of free oxygen radicals in
living organisms. Iron exists in two distinct oxidation states; ferrous (Fe
2?
) or ferric
ions (Fe
3?
). The ferric ion (Fe
3?
) is the relatively biologically inactive form of iron.
However, it can be reduced to the active Fe
2?
, depending on the conditions,
particularly pH (Strlic et al. 2002), and oxidized back through Fenton type reactions,
with production of hydroxyl radicals; or Haber–Weiss reactions with superoxide
anions (Kehrer 2000; Wong and Kitts 2001). The production of these radicals can
lead to lipid peroxidation, protein modification and DNA damage. Chelating agents
may inactivate metal ions and potentially inhibit the metal-dependent processes
(Finefrock et al. 2003).
Phenolic compounds are one of many natural chelating agents in fresh foods, in
addition to ascorbic acid, phosphorylated compounds and proteins (Gu
¨lc¸in et al.
2003b; Wong and Kitts 2001). Also, the production of highly ROS, such as
superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals is also
catalyzed by free iron through Haber and Weiss (1934). Free iron is known to have
low solubility and a chelated iron (i.e., iron-ligant) complex, such as EDTA–Fe, and
has greater solubility in solution, which can be contributed solely from the ligant.
Furthermore, chelated iron, such as EDTA–Fe, is also known to be active, since it
can participate in iron-catalyzed reactions (Wong and Kitts 2001).
Ferrous ion chelating activities of ligustroside, oleuropein, a-tocopherol, trolox,
BHA and BHT are shown in Fig. 4. The chelating effect of ferrous ions by ligustroside,
oleuropein and standards was determined according to the method of Dinis et al.
0
25
50
75
100
α-Tocopherol Trolox BHA BHT Ligustroside Oleuropein
(%)
Hydrogen peroxide scavenging Superoxide scavenging Metal chelating
Fig. 4 Comparison of hydrogen peroxide scavenging, superoxide anion radical scavenging and ferrous
ions chelating activity of ligustroside (1.85 910
-3
M) and oleuropein (1.90 910
-3
M) from root bark
of fringe tree (Chionanthus virginicus L.), BHA (5.54 910
-3
M), BHT (4.53 910
-3
M), a-tocopherol
(2.32 910
-3
M) and trolox (3.99 910
-3
M) at the same concentration (10 lg/mL) (BHA butylated
hydroxyanisole, BHT butylated hydroxytoluene)
Wood Sci Technol
123
(1994). Among the transition metals, iron is known as the most important lipid
oxidation pro-oxidant due to its high reactivity. The ferrous state of iron accelerates
lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals
via the Fenton reaction (Fe
2?
?H
2
O
2
?Fe
3?
?OH
-
?OH). Fe
3?
ion also
produces radicals from peroxides, although the rate is tenfold less than that of Fe
2?
ion
(Miller 1996). Fe
2?
ion is the most powerful pro-oxidant among various species of
metal ions (Halliwell and Gutteridge 1984).
The chelating of ferrous ions by ligustroside and oleuropein was estimated by the
ferrozine assay. Ferrozine can quantitatively form complexes with Fe
2?
. In the
presence of chelating agents, the complex formation is inhibited and the red color of
the complex fades. By measuring the color reduction, therefore, it is possible to
estimate the chelating activity of the co-existing chelator (Yamaguchi et al. 2000).
In this assay, the natural compound interfered with the formation of the ferrozine–
Fe
2?
complex, suggesting that it has chelating activity and captures ferrous ions
before ferrozine.
In fact, as shown in Fig. 4, ligustroside and oleuropein disrupted the Fe
2?
ferrozine complex at 10 lg/mL concentration. The difference among all ligustroside
and oleuropein concentrations and the control was statistically significant
(p\0.01). In addition, ligustroside (1.85 910
-3
M) and oleuropein
(1.90 910
-3
M) exhibited, respectively, 62 and 50% chelation of ferrous ion at
the above concentration. On the other hand, at the 10 lg/mL concentration, the
percentages of metal chelating capacity of BHA (5.54 910
-3
M), BHT
(4.53 910
-3
M), a-tocopherol (2.32 910
-3
M) and trolox (3.99 910
-3
M) were
found to be 72, 64 and 22 and 49%, respectively. The metal scavenging effects of
those samples decreased in the order of BHA [BHT [ligustroside [oleurop-
ein [trolox [a-tocopherol. These results showed that trolox, which is a water-
soluble synthetic analog of a-tocopherol, did not show any metal chelation activity.
Metal chelating capacity was significant, since it reduced the concentration of the
catalyzing transition metal in lipid peroxidation. The data obtained from Fig. 4
reveal that ligustroside and oleuropein demonstrate a marked capacity for iron
binding, suggesting that their main action as peroxidation protector may be related
to its iron binding capacity.
Hydrogen peroxide scavenging activity
Hydrogen peroxide can be formed in vivo by many oxidizing enzymes such as
superoxide dismutase. It can cross membranes and may slowly oxidize a number of
compounds. The ability of ligustroside and oleuropein to scavenge hydrogen peroxide,
determined according to the method of Ruch et al. (1989) is shown in Fig. 4and is
compared with that of a-tocopherol and trolox as standards. Ligustroside
(1.85 910
-3
M) and oleuropein (1.90 910
-3
M) exhibited 83 and 81% scavenging
effect of hydrogen peroxide at the 10 lg/mL concentration. On the other hand, BHA
(5.54 910
-3
M), BHT (4.53 910
-3
M), a-tocopherol (2.32 910
-3
M) and trolox
(3.99 910
-3
M) exhibited, respectively, 71, 67, 70 and 51% hydrogen peroxide
scavenging activity at the same concentration (10 lg/mL). These results showed that
ligustroside and oleuropein have effective hydrogen peroxide scavenging activity. At
Wood Sci Technol
123
the above concentration, the hydrogen peroxide scavenging effect of ligustroside,
oleuropein and four standards decreased in the order of ligustroside [oleurop-
ein [BHA [a-tocopherol [BHT [trolox. Hydrogen peroxide itself is not very
reactive; however, it can at times be toxic to cell because it may give rise to hydroxyl
radical in the cells. Addition of hydrogen peroxide to cells in culture can lead to
transition metal ion-dependent OH radicals mediated oxidative DNA damage. Thus,
removing hydrogen peroxide as well as superoxide anion is very important for
protection of pharmaceutical and food systems.
ABTS
?
radical scavenging activity
Radical scavenging activities are very important due to the deleterious role of free
radicals in foods and biological systems. Excessive formation of free radicals
accelerates the oxidation of lipids in foods and decreases food quality and consumer
acceptance (Min 1998).
The improved technique for the generation of ABTS
?
described here involves
the direct production of the blue/green ABTS
?
chromophore through reaction
between ABTS and potassium persulfate. As shown in Table 1, ligustroside and
oleuropein had ABTS
?
radical scavenging activity in a concentration-dependent
manner (10–20 lg/mL). There is a significant decrease (p\0.01) in the concen-
tration of ABTS
?
due to the scavenging capacity of ligustroside, oleuropein and
standards. In addition, the scavenging effect of ligustroside (3.70 910
-3
M),
oleuropein (3.80 910
-3
M) and standards on the ABTS
?
decreased in the order:
BHA [oleuropein [BHT [trolox [a-tocopherol [ligustroside, which were
98.0, 97.2, 96.2, 94.8, 89.2 and 51.8%, respectively, at the 20 lg/mL concentration.
DPPHradical scavenging activity
1,1-Diphenyl-2-picryl-hydrazyl free radical (DPPH) has been widely used to test
the free radical-scavenging ability of various dietary antioxidants. Antioxidants
react with DPPH, which is a stable free radical, and convert it to 1,1-diphenyl-2-
Table 1 DPPHand ABTS
?
scavenging activity of ligustroside and oleuropein from root bark of fringe
tree (Chionanthus virginicus L.), BHA, BHT, a-tocopherol and trolox
DPPHscavenging (%) ABTS
?
scavenging (%)
10 lg/mL 20 lg/mL IC
50a
R
2
10 lg/mL 20 lg/mL IC
50a
R
2
BHA 81.2 86.2 9.86 0.837 94.1 98.0 3.25 0.925
BHT 66.2 99.7 9.41 0.959 93.4 96.2 3.74 0.847
a-Tocopherol 64.9 85.2 10.62 0.901 74.9 89.7 1.57 0.982
Trolox 11.4 14.3 62.50 0.874 86.4 94.8 5.38 0.953
Ligustroside 12.0 32.8 32.2 0.972 47.0 51.8 14.69 0.861
Oleuropein 57.5 83.7 11.12 0.947 66.6 97.2 9.96 0.989
ABTS
?
2,20-Azino-bis (3-ethylbenzothiazoline-6-sulfonate) radicals, DPPH 1,1-diphenyl-2-pic-
rylhydrazyl radicals, BHA butylated hydroxyanisole, BHT butylated hydroxytoluene
a
Expressed in lg/mL concentration
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123
picryl hydrazine. The degree of discoloration indicates the radical-scavenging
potential of the antioxidant (Singh et al. 2002). Swertiamarin and sweroside,
secoiridoid glycosides, have been isolated from the aerial parts of Centaurium
erythraea and tested for DPPH free radical scavenging activities. However, neither
of the two secoiridoid glycosides showed any antioxidant activity in this assay
(Kumarasamy et al. 2003). In another work, it was found that the ethanol extract of
the fruits of Ligustrum lucidum showed significant inhibitory effects on free radical-
induced hemolysis of red blood cells. Ligustroside and oleuropein were isolated
from the active fraction of the fruits of Ligustrum lucidum (He et al. 2001). In this
study, antioxidant activities of ligustroside, oleuropein and standard antioxidants
such as a-tocopherol and trolox were determined using a DPPHmethod. Since the
DPPHassay can accommodate a large number of samples in a short period and is
sensitive enough to detect natural compounds at low concentrations, it was used in
the present study for a primary screening of the ligustroside and oleuropein free
radical-scavenging activity. This assay provides information on the reactivity of test
compounds with a stable free radical. DPPHgives a strong absorption band at
517 nm in visible spectroscopy because of its odd electron. As this electron
becomes paired off in the presence of a free radical scavenger, the absorption
vanishes, and the resulting decolorization is stoichiometric with respect to the
number of electrons taken up. Ligustroside and oleuropein exhibited marked DPPH
free radical scavenging activity in a concentration-dependent manner. Table 1
illustrates a significant decrease (p\0.05) in the concentration of DPPH radical
due to the scavenging ability of ligustroside, oleuropein and standards. BHA, BHT,
a-tocopherol and trolox were used as references radical scavengers. The scavenging
effect of ligustroside (3.70 910
-3
M), oleuropein (3.80 910
-3
M) and standards
on the DPPH radical decreased in that order: BHT [BHA [a-tocopherol [oleu-
ropein [ligustroside [trolox, which were 100, 86, 85, 84, 33 and 14%,
respectively, at the 20 lg/mL concentration.
Superoxide anion radical scavenging activity
Superoxide anion radical included in free radical species is a factor that can induce
aging and destruct the cell membrane and it can be generated by oxidative stress.
They are produced in vivo by electron leakage from the mitochondrial electron
transport chain, by activated phagocytes (Halliwell 1991) and in the conversion of
xanthenes to uric acid (Bast et al. 1991). Superoxide anions are precursor to active
free radicals that have the potential to react with biological macromolecules, thereby
inducing tissue damage (Halliwell and Gutteridge 1984). Also, it has been
implicated in several pathophysiological processes due to its transformation into
more reactive species, such as hydroxyl radical that initiate lipid peroxidation.
Superoxide has also been observed to directly initiate lipid peroxidation (Wickens
2001). It has also been reported that antioxidant properties of some flavonoids are
effective, mainly via scavenging of superoxide anion radical (Yen and Duh 1994).
Superoxide anion plays an important role in formation of other ROS such as
hydrogen peroxide, hydroxyl radical, and singlet oxygen, which induce oxidative
damage in lipids, proteins, and DNA (Pietta 2000). Also, superoxide anion is an
Wood Sci Technol
123
oxygen-centered radical with selective reactivity. This species is produced by a
number of enzyme systems in auto-oxidation reactions and by non-enzymatic
electron transfers that univalently reduce molecular oxygen. It can also reduce
certain iron complex such as cytochrome c.
In this method, superoxide anion derived from dissolved oxygen by PMS–NADH
coupling reaction reduces the yellow dye (NBT
2?
) to produce the blue formazan,
which is measured spectrophotometrically at 560 nm. Antioxidants are able to
inhibit the blue NBT formation (Cos et al. 1998; Parejo et al. 2002). The decrease of
absorbance at 560 nm with antioxidants indicates the consumption of superoxide
anion in the reaction mixture. Figure 4shows that the percentage inhibition of
superoxide radical generation by 10 lg/mL concentration of ligustroside; oleurop-
ein and standards were found to be statistically similar. As can be seen in Fig. 4, the
percentage inhibition of superoxide anion radical generation by 10 lg/mL
concentration of ligustroside (1.85 910
-3
M) and oleuropein (1.90 910
-3
M)
was found to be 69 and 34%. On the other hand, at the same concentration, BHA
(5.54 910
-3
M), BHT (4.53 910
-3
M), a-tocopherol (2.32 910
-3
M) and
trolox (3.99 910
-3
M) exhibited 76, 47, 71 and 78% superoxide anion radical
scavenging activity, respectively.
Conclusion
This study demonstrated the potential antioxidant properties of two secoiridoids,
ligustroside and oleuropein, from fringe tree (Chionanthus virginicus). Oleuropein
has an extra phenolic hydroxyl group compared to ligustroside. Because of this
phenolic hydroxyl group, it was respected that oleuropein has higher antioxidant
activity than ligustroside. We found similar results in another study. In that study, it
was found that L-dopa had higher antioxidant activity than L-tyrosine because of
extra hydroxyl group (Gu
¨lc¸in 2007). According to data of the present study,
ligustroside and oleuropein were found to be effective antioxidants in different in
vitro assays including ferric thiocyanate method, reducing power, DPPHscaveng-
ing, ABTS
?
scavenging and superoxide anion radical scavenging, hydrogen
peroxide scavenging and metal chelating activities when compared to standard
antioxidant compounds, such as synthetic antioxidants (BHA, BHT), a-tocopherol, a
natural antioxidant, and trolox, which is a water-soluble analog of tocopherol.
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... The ferric reducing antioxidant power (FRAP) assay is a simple test that uses a reductant in a redox reaction with an oxidant and is combined with a colorimetric method [53]. The addition of Fe 3+ to the reduced product by addition of phyto and mammalian lignans leads to the formation of Fe4[Fe(CN)6], a complex in the Prussian blue color with sharp absorbance at 700 nm [54,55]. ...
... The ferric reducing antioxidant power (FRAP) assay is a simple test that uses a reductant in a redox reaction with an oxidant and is combined with a colorimetric method [53]. The addition of Fe 3+ to the reduced product by addition of phyto and mammalian lignans leads to the formation of Fe 4 [Fe(CN) 6 ], a complex in the Prussian blue color with sharp absorbance at 700 nm [54,55]. ...
Article
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In this study, the antioxidant and antiradical properties of some phyto lignans (nordihydroguaiaretic acid, secoisolariciresinol, secoisolariciresinol diglycoside, and α-(-)-conidendrin) and mammalian lignans (enterodiol and enterolactone) were examined by different antioxidant assays. For this purpose, radical scavenging activities of phyto and mammalian lignans were realized by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical (ABTS•+) scavenging assay and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging assay. Additionally, the reducing ability of phyto and mammalian lignans were evaluated by cupric ions (Cu2+) reducing (CUPRAC) ability, and ferric ions (Fe3+) and [Fe3+-(TPTZ)2]3+ complex reducing (FRAP) abilities. Also, half maximal inhibitory concentration (IC50) values were determined and reported for DPPH• and ABTS•+ scavenging influences of all of the lignan molecules. The absorbances of the lignans were found in the range of 0.150–2.320 for Fe3+ reducing, in the range of 0.040–2.090 for Cu2+ reducing, and in the range of 0.360–1.810 for the FRAP assay. On the other hand, the IC50 values of phyto and mammalian lignans were determined in the ranges of 6.601–932.167 µg/mL for DPPH• scavenging and 13.007–27.829 µg/mL for ABTS•+ scavenging. In all of the used bioanalytical methods, phyto lignans, as secondary metabolites in plants, demonstrated considerably higher antioxidant activity compared to that of mammalian lignans. In addition, it was observed that enterodiol and enterolactone exhibited relatively weaker antioxidant activities when compared to phyto lignans or standard antioxidants, including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), Trolox, and α-tocopherol.
... Food alterations may occur during the production of foods through to consumption, during the storage of living raw materials or the storage of finished products. A number of physical (colour change), chemical (oxidation), biochemical (destruction of vitamins) and microbiological alterations (fermentation, development of pathogenic microorganisms, and production of toxins) may occur [426,427]. A wide range of food products, such as oil-in-water emulsions (milk, cream, mayonnaise, margarine, etc.) and meat products (sausages and patties) are easily exposed to deterioration during processing and conservation steps, highlighting the importance of their analysis to find effective solutions and extend their shelf life [405]. ...
... Chemical structure of the butylated hydroxytoluene (BHT)[427] ...
Thesis
The present work focused on the study of two edible plants, with attributable medicinal values, named Anthyllis vulneraria L. (A.vulneraria) and Azadirachta indica L. (A.indica) collected in Tunisia and India, respectively. The aim of the work was to determine the chemical composition of A.vulneraria leaf and flower extracts and A.indica leaf extract and to investigate their antioxidant, antibacterial and anti-proliferative (against specific cancer cell lines) activities in order to design formulations for use in the food and cosmetic industries. The chemical composition of the extracts was estimated by spectrophotometric assays and quantified by High Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS) and diode array (HPLC-DAD). The radical scavenging activity of the extracts was evaluated in vitro by different analytical methods. The antioxidant capacity of A. vulneraria and A. indica samples in oil-in-water emulsions and in raw minced beef was also studied to evaluate their preservative and quality evolution effect, in general, and the shelf life of these food products during the storage period. In addition, the antibacterial activity of the different extracts was tested against pathogenic microorganisms causing food poisoning and the anti-proliferative activity was evaluated against several human cancer cell lines. The results obtained revealed that: (1) aqueous EtOH extracts of A.vulneraria and A.indica showed better extraction yields, higher content of phenolic compounds and higher anti-radical activity than pure EtOH extracts. (2) A. vulneraria flowers showed higher biological activities than leaves. (3) In Model Food System, at 0.25% (v/v), A.vulneraria flowers extracted in 50%-aqueous EtOH protected oil-in-water emulsions against oxidation and prolonged shelf life better than leaf extract and even better than the sample with gallic acid (under the same conditions). Moreover, freeze-dried A.vulneraria flower sample, added at 0.5% (w/w) to minced beef, showed better results in protecting against lipid oxidation and in maintaining its organoleptic properties than freeze-dried leaf sample. The preservative effect of freeze-dried A. vulneraria leaf and flower was slightly lower than that of the synthetic preservative (0.5%, w/w). Furthermore, the combination of A. indica and Capsicum baccatum showed a strong synergistic effect against the spoilage of meat products even better than the synthetic preservative (0.7%, w/w). (4) In the study of the antibacterial activity of the extracts, it was found that, on the one hand, the flower extract of A. vulneraria (100 µg/mL) showed better inhibition of the growth of most of the tested bacterial strains than leaf extract. On the other hand, leaf extract of A. indica (100 µg/mL) had better antibacterial activity than penicillin. (5) The study of the anti-proliferative activity of extracts against human cancer cells showed that, at 1% (v/v), A. vulneraria leaf extract was more effective in preventing the proliferation of cancer cells than A.vulneraria flower extract, especially HepG2 cells. At 5% (v/v), both extracts (leaf and flower) showed potent anti-proliferative activity against the three cancer cell lines tested. Moreover, A. indica leaf extract at 7% (v/v) showed a more potent reduction of breast and cervical adenocarcinoma-derived cell lines viability (MCF-7 and HeLa) than hepatocellular carcinoma derived cells (HepG2). Different compounds that could be responsible for the antioxidant, antibacterial and anti-proliferative activities of the different extracts investigated, belonging to the phenolic acid and flavonoid families, were identified and quantified by HPLC-MS. Keywords: Anthyllis vulneraria, antibacterial activity, anti-proliferative activity, Azadirachta indica, emulsion, HPLC-MS, meat products, oxidative stability, radical scavenging activity, synergistic effect.
... Antioxidants block the harmful effects of ROS and are divided into two main groups, natural and synthetic, prevent free radicals from harming the body by catching and neutralizing them [88][89][90]. Antioxidants are described as substances that effectively scavenge ROS, positively regulate antioxidant defense systems or inhibit ROS production [91,92]. ...
Article
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Heavy metals are essential for a wide range of biological processes, including the growth and reproduction of cells, synthesis of biomolecules, many enzymatic reactions, and the body’s immunity, but their excessive intake is harmful. Specifically, they cause oxidative stress (OS) and generate free radicals and reactive oxygen species (ROS) in metabolism. In addition, the accumulation of heavy metals in humans can cause serious damage to different organs, especially respiratory, nervous and reproductive and digestive systems. Biologically, metal chelation therapy is often used to treat metal toxicity. This process occurs through the interaction between the ligand and a central metal atom, forming a complex ring-like structure. After metals are chelated with appropriate chelating agents, their damage in metabolism can be prevented and efficiently removed from the body. On the other hand, heavy metals, including Zn, Fe and Cu, are necessary for the suitable functioning of different proteins including enzymes in metabolism. However, when the same metals accumulate at levels higher than the optimum level, they can easily become toxic and have harmful effects toward biomolecules. In this case, it induces the formation of ROS and nitrogen species (RNS) resulting in peroxidation of biological molecules such as lipids in the plasma membrane. Antioxidants have an increasing interest in many fields due to their protective effects, especially in food and pharmaceutical products. Screening of antioxidant properties of compounds needs appropriate methods including metal chelating assay. In this study, a general approach to the bonding and chelating properties of metals is described. For this purpose, the basic principles and chemical principles of metal chelation methods, both in vivo and in vitro, are outlined and discussed. Hence, in the main sections of this review, the descriptions related to metal ions, metal chelating, antioxidants, importance of metal chelating in biological system and definitions of metal chelating assays as widely used methods to determine antioxidant ability of compounds are provided. In addition, some chemical properties, technical and critical details of the used chelation methods are given.
... The metal-chelating capacity of Schiff bases (9a-c), thiazolidinones (10a-c) and azetidinones (11a-c) was estimated adapting Dinis et al. procedure [50] with some modifications [51,52]. Fe 2+ binding ability of 9a-c, 10a-c and β -lactam analogs (11a-c) had been recorded spectrophotometrically at 562 nm. ...
Article
Background & objectives 1,2-thiazine and pyridine heterocycles drew much attention due to their biological activities including antioxidant activity. Based upon fragment based drug design, novel pyrido[1,2]thiazines 9a-c, thiazolidinopyrido[1,2]thiazines 10a-c and azetidinopyrido[1,2]thiazines 11a-c were designed and prepared. Methods These novel derivatives 9a-c, 10a-c and 11a-c were subjected to screening for their antioxidant activity via various assays as DPPH radical scavenging potential, reducing power assay and metal chelating potential. Results All the assayed derivatives exhibited excellent antioxidant potential and the tested compounds 9a, 9b, 10a, 10b, 11a and 11b exhibited higher DPPH scavenging potential (EC50 = 32.7, 53, 36.1, 60, 40.6 and 67 µM, respectively) than ascorbic acid (EC50 = 86.58 µM). While targets 9a, 10a and 11a (RP50 = 52.19, 59.16 and 52.25 µM, respectively) exhibited better reducing power than the ascorbic acid (RP50 = 84.66 µM). Computational analysis had been utilized to prophesy the bioactivity and molecular properties of the target compounds. Conclusion To predict the binding manner of the novel derivatives as antioxidants, in-silico docking study had been performed to all the newly prepared compounds inside superoxide dismutase (SOD) and catalase (CAT) active site. The most active antioxidant candidate 9a (EC50 = 32.7 µM, RP50 = 52.19 µM) displayed excellent binding with Lys134 amino acid residing at Cu-Zn loop of SOD with binding energy score = -7.54 Kcal/mol thereby increase SOD activity and decrease reactive oxygen species.
... Furthermore, ROSs, which have been linked to a variety of illnesses, are created by all living cells as a fundamental immunological defense mechanism [34,35]. Oxidative stress and Reactive Oxygen Species (ROS) have recently been identified as substantial environmental dangers for a variety of chronic diseases, including tumors, aids syndrome, age-related pathologies, cardiovascular disease, arteriosclerosis, diabetes, and obesity [36,37]. Antioxidant components and an antioxidant enzyme make up the antioxidant defense system (Figure 3). ...
Article
Full-text available
Alzheimer’s disease is a neurodegenerative illness marked by a gradual memory impairment and certain intellectual (neurocognitive) functions leading to repercussions in the activities of daily living. Until now, there is no drug to treat neurodegenerative disorders; for this, it is preferable to seek to delay the progression of this disease. Lichens show vital therapeutic activity in several neurological diseases, including Alzheimer’s disease. Several isolated lichenic compounds have been tested for anti-acetylcholinesterase potency and may play a key role in the prevention of this dementia. This review deals with previous work on the therapeutic activity of some lichens and their bioactive components for them neurodegenerative diseases. Thus, compounds isolated from lichens can be considered favorable and promising for the prevention of neurodegenerative diseases.
... Structure chimique de l'hydroxytoluène butylé (BHT)[440]. ...
Thesis
Cette thèse a porté sur l’analyse phytochimique de l’Anthyllis vulneraria (Fabaceae) et l’Azadirachta indica (Meliaceae) collectées en Tunisie et en Inde, respectivement et la détermination de leurs activités antioxydantes, antibactériennes et antiprolifératives afin de concevoir des formulations pour les utiliser dans l’industrie alimentaire, pharmaceutique et cosmétique. L’étude de la composition chimique des extraits par spectrophotométrie et par HPLC-MS a montré que l’extrait foliaire et l’extrait floral de l’A. vulneraria et l’extrait foliaire de l’A. indica sont riches en acides phénoliques et flavonoïdes à des teneurs différentes. L’évaluation du pouvoir antioxydant a montré que les différents extraits ont une forte activité anti-radicalaire à faible concentration et possèdent un fort pouvoir conservateur sur la stabilité oxydative des matrices alimentaires; y compris l’émulsion huile dans l’eau et les produits carnés, similaire voire meilleur que le conservateur synthétique. En plus, l’étude de l’activité antibacterienne des différents extraits montre que l’extrait foliaire de l’A. indica a une forte activité antibacterienne contre toutes les souches bactériennes testées sauf la Listeria monocytogenes et Bacillus cereus, et que l’extrait floral de l’A. vulneraria a un pouvoir inhibiteur de la croissance bactérienne plus fort que l’extrait foliaire. L’étude de l’activité antiproliferative des extraits montre que l’extrait foliaire de l’A. vulneraria a réduit la viabilité des lignées cellulaires cancéreuse mieux que l’extrait floral surtout la HepG2 alors que l’extrait foliaire de l’A. indica a eu un effet antiproliferative plus fort contre la HeLa et MCF-7 que la HepG2. En résumé, l’A. vulneraria et l’A. indica ont des propriétés biologiques pertinentes du point de vue de la santé et représentent un grand potentiel en tant qu'additif alimentaire permettant la conservation des produits alimentaires et l'amélioration de leurs valeurs nutritionnelles. Mots clés: activité antibactérienne, activité antiproliférative, activité anti-radicalaire, Anthyllis vulneraria, Azadirachta indica, émulsion, HPLC-MS, produits carnés, stabilité oxydative.
Article
In this study, some biochemical properties of Satureja avromanica such as phenolic content, anticholinergic, antidiabetic and antioxidant activities were determined. Ethanol extract of Satureja avromanica (EESA) and water extract of Satureja avromanica (WESA) were prepared and used for all biochemical analyses. The antioxidant capacities of EESA and WESA were evaluated by six different antioxidant methods. In addition, acetylcholinesterase (AChE), α-amylase and α-glycosidase enzyme inhibition of EESA were measured. EESA exhibited high inhibition effects against α-amylase, α-glycosidase and AChE enzymes. The IC50 values of EESA against AChE (1.909 μg/mL), α-glycosidase (0.701 μg/mL), and α-amylase (0.517 μg/mL) were determined. The phenolic composition of Satureja avromanica was evaluated by LC-HRMS. Rosmarinic acid (19961.31 mg/kg), hederagenin (18895.43 mg/kg) and hesperidin (11409.60 mg/kg) were identified as major compounds in EESA. Rosmarinic acid (9209.82 mg/kg), hesperidin (2384.73 mg/kg) and hispidulin (1530.03 mg/kg) were identified as major compounds in WESA. Molecular docking analysis showed possible roles of hesperidin, hederagenin, and rutin in AChE, α-amylase and α-glycosidase enzymes inhibition. Also, the highly cytotoxic effect of EESA, which is thought to be caused by phenolic compounds, was observed in A495 cancer cells.
Article
A new coumarin derivative, 7-O-methylparamicoumarin A (1), has been isolated from the roots of Chionanthus retusus, together with seven known compounds. The structure of new compound 1 was determined through spectroscopic and MS analyses. Among the isolates, luteolin (3), quercetin (4), and apigenin (5) exhibited cytotoxicities with IC50 values of 13.69 ± 1.10, 10.41 ± 0.79, and 12.25 ± 0.91 μM, respectively, against DLD-1 cell line. Luteolin (3), quercetin (4), and apigenin (5) also exhibited cytotoxicities with IC50 values of 15.35 ± 1.24, 13.52 ± 0.87, and 19.48 ± 1.26 μM, respectively, against CCRF-CEM cell line. In addition, kaempferol (2), apigenin (5), 3,3′,5,5′,7-pentahydroxyflavanone (6), and oleuropein (8) showed potent inhibition with IC50 values of 24.84 ± 1.95, 27.18 ± 1.82, 25.18 ± 2.07, and 28.14 ± 1.66 μM, respectively, against LPS-induced NO generation.
Article
The antioxidant activity and antibacterial efficacy of aqueous and ethanolic extracts of Chaetomorpha linum were tested against bacterial infection caused by Pseudomonas aeruginosa. In vitro and in vivo studies revealed the efficacy of ethanolic extract of C. linum in controlling the P. aeruginosa infection. Gas Chromatography Mass Spectroscopy analysis of purified fractions of ethanolic extract was also performed. Ichthyotoxic bioassay of purified seaweed extracts was performed to assess its toxic effect in rohu fish and found to be nontoxic. Experimental pathogenicity of P. aeruginosa showed exophthalmia, reduced intake of feed, and lesions on the body. LD 50 was achieved at the 3.96 × 102 at 48 h and 4.82 × 103 at 72 h in the experimental pathogenicity. The results revealed that the ethanolic extract of C. linum can be used as an alternate source for controlling P. aeruginosa infection in L. rohita.
Article
The growing need for biomass recovery suggests forest waste leaf material for technological applications in a circular economy scenario. In this context, white poplar (Populus alba L.) foliar material was recovered in a forest site planted on a former agricultural land was identified in Tuscany (Italy), and intercropping eventually occurred was also valuated. In fact, the mixed plantation was characterized by tree different associations consisting of broad-leaf trees, including Populus alba L. intercropped with another valuable species (walnut, Juglans regia L.), and different nurse species (Italian alder, Alnus cordata (Loisel.); hazelnut, Corylus avellana L., Autumn olive, Elaeagnus umbellata (Thunb.)). Thus, Populus albaleaves were investigated for their lignin and phenol content, and for their anti-radical activity by (2,2-diphenyl-1-picrylhydrazyl) DPPH and [2, 2-azinobis (3-eth-ylbenzothiazoline-6-sulfonic acid)] ABTS assays. Furthermore, Populus alba extracts were profiled by liquid chromatography hyphenated to high-resolution tandem mass spectrometry (LC-HRMS/MS), in order to deepen into the intercropping influence on specialized metabolites' content. In particular, it was observed that when Populus alba grows in presence of the nurse species Elaeagnus umbellata, a decrease in the aforementioned parameters was observed, as well as a negative impact on the polyphenol profile. Thus, our findings are in line with the observation that white poplar leaf residue has a high potential for achieving bioactive polyphenol compounds , and that an intercropped nurse species such as Alnus cordata could favourably augment flavonoids and chlorogenic acids to be used as multifunctional ingredients.
Article
Antioxidative activity of aromatic amino acids and indole compounds for the autoxidation of linoleic acid was found to correlate in some extent with the highest occupied molecular orbital energy which represents the electron donor property of respective molecule. 5-Hydroxytryptophan, one of the best electron donor among the compounds tested, was the most effective antioxidant. However, antioxidative activity of some indole compounds could not be interpreted simply by their highest molecular orbital energies.Neither the chelating action for the possible metal traces nor the accelerated decomposition of hydroperoxide produced during the course of the reaction explained these actions of indoles. Tryptophan, while preventing the autoxidation of linoleic acid, underwent the ring cleavage at the position of between C2 and C3 or hydroxylation at C5 to yield formylkynurenine, kynurenine, 3-hydroxykynurenine, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, etc. Following mechanisms which were compatible with the experimental results were proposed for the antioxidative action of indoles; indole donates an electron from its π-pool to linoleic acid radical or peroxy radical produced during the autoxidation of linoleic acid to form a loose charge transfer complex through a “local” interaction; an electron transfer occurs within the complex, which brings cleavage of indole rings and an inhibition of autoxidation.