Content uploaded by Ganiyu Oboh
Author content
All content in this area was uploaded by Ganiyu Oboh
Content may be subject to copyright.
Polyphenols in red pepper [Capsicum annuum var. aviculare
(Tepin)] and their protective effect on some pro-oxidants
induced lipid peroxidation in brain and liver
Ganiyu Oboh ·Joao Batista T. Rocha
Received: 10 March 2006 / Revised: 9 June 2006 / Accepted: 11 June 2006 / Published online: 30 August 2006
C
Springer-Verlag 2006
Abstract Polyphenols exhibit a wide range of biological
effects because of their antioxidant properties. The study
sought to carry out a comparative studies on the protective
ability of free and bound polyphenol extracts of red Cap-
sicum annuum var. aviculare (Tepin) on brain and liver – in
vitro. Free polyphenols of red Capsicum annuum var. avic-
ulare (Tepin) were extracted with 80% acetone, while the
bound polyphenols were extracted with ethyl acetate from
acid and alkaline hydrolysed residue from free polyphe-
nols extract. The phenol content, Fe(II) chelating ability,
OH radical scavenging ability and protective ability of the
extract against some pro-oxidant (25 µM Fe(II), 7 µM
sodium nitroprusside and 1 mM quinolinic acid)-induced
lipid peroxidation in brain and liver was subsequently de-
termined. The results of the study revealed that the free
polyphenols (218.2 mg/100 g) content of the pepper were
significantly higher (P<0.05) than the bound polyphenols
(42.5 mg/100 g). Furthermore, the free polyphenol extract
had a significantly higher (<0.05) Fe(II) chelating ability,
OH radical scavenging ability than the bound polyphenols. In
addition, both extracts significantly inhibited (P<0.05) basal
and the various pro-oxidant (25 µM Fe(II), 7 µM sodium
nitroprusside and 1 mM quinolinic acid)-induced lipid per-
oxidation in rat’s brain and liver in a dose-dependent man-
ner. However, the free polyphenols caused a significantly
G. Oboh ()
Biochemistry Department, Federal University of Technology,
P.M.B. 704 Akure, Nigeria
e-mail: goboh2001@yahoo.com
Tel.: +2348035600192
G. Oboh ·J. B. T. Rocha
Departamento de Quimica, Universidade Federal de Santa Maria
(UFSM), Campus Universit´
ario – Camobi,
97105-900 Santa Maria RS, Brazil
higher inhibition in the MDA (malondialdehyde) produc-
tion in the brain and liver homogenates than the bound phe-
nols. In conclusion, Capsicum annuum var. aviculare (Tepin)
contains 83.7% free soluble polyphenol and 16.3% bound
polyphenols. In addition, both polyphenolic extracts inhibit
the various pro-oxidant agents (Fe2+, sodium nitroprusside
and quinolinic acid) induced lipid peroxidation in brain and
liver tissues in a doe-dependent manner. However, the free
polyphenols had higher protective ability than the bound
polyphenols. The main mechanism through which they are
carry out their protective effect against lipid peroxidation
in the brain and the liver is by Fe(II) chelating ability, OH
and NO radicals scavenging ability and inhibition of over-
stimulation of NMDA receptor.
Keywords Polyphenols .Capsicum annuum var. aviculare
(Tepin) .Fe(II) .Lipid peroxidation
Introduction
Lipid peroxidation in biological membranes has been con-
sidered as one of the major mechanisms of cell injury in aero-
bic organisms subjected to oxidation stress [1,2]. Hochstein
et al. discovered the requirement for iron in initiating lipid
peroxidation in the 1960s [3]. Since then, the mechanism
involved in iron-dependent lipid peroxidation has been stud-
ied in many in vitro model systems, such as liposomes and
microsomes [4].
Although Fe is necessary in relatively large amounts for
hemoglobin, myoglobin and cytochrome production, xan-
thine oxidase and the other Fe proteins require rather small
amounts of Fe. On the other hand, free Fe in the cytosol and
in the mitochondria can cause considerable oxidative dam-
age by increasing superoxide production. Through Fenton
reactions and by activating xanthine oxidase, which pro-
duces both uric acid (an antioxidant that recycles ascorbic
acid in the cell and is therefore vital to the animals that do
not produce ascorbic acid, such as primates) and O2∗, which
causes massive damage either by itself or by reacting with ni-
tric oxide (NO) to form the powerful peroxynitrite (ONOO∗)
[5].
High levels of both Cu and Fe, with relatively low levels
of Zn and Mn play a crucial role in brain cancer and in de-
generative diseases of the brain (Parkinson’s and Alzheimer’s
diseases, multiple sclerosis, etc.) [5]. Because of iron storage
disease, the liver becomes cirrhotic. Hepatoma, the primary
cancer of the liver, has become the most common cause of
death among patients with hemochromatosis. In addition,
when siderosis becomes severe in young people, myocardial
disease is a common cause of death. Sodium nitroprusside
is an anti-hypertensive drug, it acts by relaxation of vascular
smooth muscle; consequently, it dilates peripheral arteries
and veins. However, sodium nitroprusside (SNP) has been
reported to cause cytotoxicity through the release of cyanide
and/or nitric oxide (NO). NO is a free radical with short half-
life (<30 s). Although NO acts independently, it also may
cause neuronal damage in cooperation with other reactive
oxygen species (ROS) [6].
Quinolinic acid (QA) is a neuroactive metabolite of the
tryptophan–kinurenine pathway, which can be produced by
macrophages and microglia. It is present in both the human
and rat brain and it has been implicated in the pathogenesis of
a variety of human neurological diseases. Quinolinic acid is
recognized pharmacologically as an endogenous glutamate
agonist with a relative selectivity for the NMDA receptor in
the brain. Since it is not readily metabolized in the synaptic
cleft, it stimulates the NMDA receptor for prolonged peri-
ods. This sustained stimulation results in opening of calcium
channels causing Ca2+influx followed by Ca2+-dependent
enhancement of free radical production leading to molecular
damage and often to cell death [7].
Phenolic compounds are an important group of secondary
metabolites, which are synthesized by plants because of plant
adaptation to biotic and abiotic stress conditions (infection,
wounding, water stress, cold stress, high visible light). Pro-
tective phenylpropanoid metabolism in plants has been well
documented [8–10]. In recent years, phenolic compounds
have attracted the interest of researchers because they show
promise of being powerful antioxidants that can protect the
human body from free radicals, the formation of which is
associated with the normal natural metabolism of aerobic
cells [11,12]. The antiradical activity of flavonoids and phe-
nolics is principally based on the redox properties of their
hydroxyl groups and the structural relationships between
different parts of their chemical structure [13–15]. Epidemi-
ological data have indicated beneficial effects of antioxidant
compounds in the prevention of a multitude of disease states,
including cancer, cardiovascular disease, and neurodegener-
ative disorders [16,17].
Compelling evidence has led to the conclusion that
diet is a key environmental factor and a potential tool
for the control of chronic diseases. After tobacco, inade-
quate diet and activity patterns are the most prominent con-
tributors to mortality in USA [18]. Dietary recommenda-
tions for the prevention of cancer, atherosclerosis and other
chronic diseases have been established by various health
agencies [19,20]. More specifically, fruits and vegetables
have been shown to exert a protective effect [21–24]. The
high content of polyphenol antioxidants in fruits and veg-
etables is probably the main factor responsible for these
effects.
Food going through the human gastrointestinal tract is
digested in the stomach (strong acid environment with en-
zymes), small intestine (mild base environment with en-
zymes), and then colon (neutral pH with intestinal mi-
croflora). Phenolics in vegetables are present in both free
and bound forms. Bound phenolics, mainly in the form of β-
glycosides, may survive human stomach and small intestine
digestion and reach the colon intact, where they are released
and exert their bioactivity [25].
Capsicum annuum var. aviculare (Tepin) is the original
wild chile – the plant from which all others have evolved. It
is a tiny round berry slightly larger than a peppercorn. It is
very decorative and bright scarlet in colour and, despite its
high heat level, it is attractive to wild birds, which helped to
distribute it across the prehistoric Americas. Our preliminary
data revealed that the pepper have high phenolic content
and antioxidant activity [26]. This study therefore sought to
investigate phenolic distribution and compare their protective
ability against some pro-oxidant induced lipid peroxidation
in rat’s brain and liver.
Materials and methods
Materials
Ripe Capsicum annuum var. aviculare (Tepin) was collected
from a vegetable garden in Camobi, Santa Maria RS, Brazil.
The authentication of the pepper was carried out in Depar-
tamento de Biologia, Universidade Federal de Santa Maria,
Santa Maria RS, Brazil. All the chemicals used were analyt-
ical grade, while the water was glass distilled. The handling
and the use of the animal were in accordance with NIH Guide
for the care and use of laboratory animals. In the experiments
Wister strain albino rats weighing 200–230 g were collected
from the breeding colony of Departamento de Biologia, Uni-
versidade Federal de Santa Maria, Santa Maria RS, Brazil.
They were maintained at 25 ◦C, on a 12 h light/12 h dark
cycle, with free access to food and water.
Extraction of soluble free phenolic compounds
For the extraction of soluble free phenols, 100 g of the pepper
was homogenized in 80% acetone (1:2 w/v) using chilled
Waring blender for 5 min. The sample was homogenized
further using a Polytron homogenizer for an additional 3 min
to obtain a thoroughly homogenized sample. Thereafter, the
homogenates were filtered through Whatman No. 2 filter
paper on a Buchner funnel under vacuum. The residues kept
for extractions of bound phytochemicals. The filtrate was
evaporated using a rotary evaporator under vacuum at 45 ◦C
until ∼90% of the filtrate had been evaporated. The extracts
were frozen at −40 ◦C[27].
Extraction of bound phenolic compounds
The residues from above soluble free extraction were drained
off and hydrolyzed directly with 20 ml of 4 M NaOH at room
temperature for 1 h with shaking. The mixture was acidified
to pH 2 with concentrated hydrochloric acid and extracted
six times with ethyl acetate. The ethyl acetate fraction was
later evaporated at 45 ◦C under vacuum to dryness [27].
Preparation of tissue homogenates
The rats were decapitated under mild diethyl ether anaes-
thesia and the cerebral tissue (whole brain) and liver were
rapidly dissected and placed on ice and weighed. These tis-
sues were subsequently homogenized in cold saline (1/10
w/v) with about 10 up-and-down strokes at approximately
1200 rev/min in a Teflon-glass homogenizer. The ho-
mogenate was centrifuge for 10 min at 3000×gto yield
a pellet that was discarded and a low-speed supernatant (S1)
was kept for lipid peroxidation assay [28].
Lipid peroxidation and thiobarbibutric acid reactions
The lipid peroxidation assay carried out using the modified
method of Ohkawa et al. [29], briefly 100 µl S1 fraction was
mixed with a reaction mixture containing 30 µlof0.1M
pH 7.4 Tris-HCl buffer, pepper extract (0–100 µl) and 30 µl
of the pro-oxidant(250 µM freshly prepared FeSO4,70 µM
sodium nitroprusside and 10 mM quinolinic acid) and the
volume was made up to 300 µl by water before incubation at
37 ◦C for 1 h. The colour reaction was developed by adding
300 µl 8.1% SDS (sodium doudecyl sulphate) to the reaction
mixture containing S1, this was subsequently followed by the
addition of 600 µl of acetic acid/HCl (pH 3.4) mixture and
600 µl 0.8% TBA (thiobarbituric acid). This mixture was
incubated at 100 ◦C for 1 h. TBARS (thiobarbituric acid
reactive species) produced were measured at 532 nm and the
absorbance was compared with that of standard curve using
MDA (malondialdehyde).
Total phenol determination
The total phenol content was determined by mixing 0.5 ml of
the extract of the pepper with 2.5 ml 10% Folin-Cioalteu’s
reagent (v/v) and 2.0 ml of 7.5% Sodium carbonate was
subsequently added. The reaction mixture was incubated at
45 ◦C for 40 min, and the absorbance was measured at 765 nm
in the spectrophotometer, gallic acid was used as standard
phenol [30].
Fe2+chelation assay
The ability of the aqueous extract to chelate Fe2+was de-
termined using a modified method of Minotti and Aust [31]
with a slight modification by Puntel et al. [32]. Briefly 150 µl
of freshly prepared 500 µM FeSO4was added to a reaction
mixture containing 168 µl of 0.1 M Tris-HCl (pH 7.4), 218 µl
saline and the aqueous extract of the pepper (0–25 µl). The
reaction mixture was incubated for 5 min, before the ad-
dition of 13 µl of 0.25% 1,10-phenanthroline (w/v). The
absorbance was subsequently measured at 510 nm in the
spectrophotometer.
Hydroxyl radical scavenging ability
The ability of the aqueous extract of the pepper to prevent
Fe2+/H2O2induced decomposition of deoxyribose was car-
ried out using the method of Halliwell and Gutteridge [33].
Briefly, freshly prepared aqueous extract (0–100 µl) was
added to a reaction mixture containing 120 µl 20 mM de-
oxyribose, 400 µl 0.1 M phosphate buffer, 40 µl20mMhy-
drogen peroxide and 40 µl 500 µM FeSO4, and the volume
for made to 800 µl with distilled water. The reaction mixture
was incubated at 37 ◦C for 30 min, and the reaction was
stop by the addition of 0.5 ml of 2.8% TCA (trichloroacetic
acid), this was followed by the addition of 0.4 ml of 0.6%
TBA solution. The tubes were subsequently incubated in
boiling water for 20 min. The absorbance was measured at
measured at 532 nm in spectrophotometer.
Analysis of data
The result of the replicates were pooled and expressed as
mean±standard error (SE) [34]. A one-way analysis of vari-
ance (ANOVA) and the least significance difference (LSD)
were carried out. Significance was accepted at P≤0.05.
Results and discussion
Most of the previous research on the phenolic contents of
plant foods determined mainly the soluble free phenolics
based on solvent-soluble extraction. However, recent reports
Table 1 Phenolic content of Red Capsicum annuum var. aviculare
(Tepin) extract
Sample Total phenol (mg/100 g) Percentage (%)
Free 218.2±2.1 a 83.7
Bound 42.5±2.8 b 16.3
Values represent means of triplicate.
Values with the same alphabet along the same column are not signifi-
cantly different (P>0.05).
as shown that in addition to the soluble free phenolics, there
are bound phenolics, which exist mainly in the form of β-
glycosides that are usually release and absorb in the colon
[27,35]. This study provides information on the phenolics
content in hot red pepper [Capsicum annuum var. aviculare
(Tepin)] and their protective ability on some pro-oxidant
induced lipid peroxidation in rat’s brain and liver.
The results of the phenolics content in ripe Cap-
sicum annuum var. aviculare (Tepin) is presented in
Table 1. The results revealed that the free phenolic con-
tents (218.2 mg/100 g) were significantly higher (P<0.05)
than the bound phenolics content (42.5 mg/100 g) in the
red pepper . These results agree with earlier reports by Chu
et al. [27] and Sun et al. [35]. However, the soluble free
phenolics content of red Capsicum annuum var. aviculare
(Tepin) was significantly higher (P<0.05) than the soluble
free phenolic content of red pepper, potato, lettuce, cucum-
ber, carrot, onion, spinach and broccoli reported by Chu et al.
[27]; nevertheless, it is lower than the soluble free phenolics
compounds in cranberry and apple [35]; nevertheless, it is
within the same range with that of red grape [35]. However,
the bound phenolics compound of the pepper was generally
higher than that of broccoli, cucumber, grape, onion, apple,
red pepper [27,35].
The percentage phenolics content in the pepper is also
presented in Table 1. The results revealed that Capsicum
annuum var. aviculare (Tepin) contains 83.7% free soluble
phenolics and 16.3% bound phenolics compound, the ratio
of free soluble phenolics to bound phenolics is lower than
the ratio that Chu et al. [27] reported free to bound pheno-
lics in spinach, red pepper and onion, but higher than that
of broccoli, cabbage, carrot, celery, cucumber, lettuce [27].
Free phenolics are more readily absorbed and thus, exert
beneficial bioactivities in early digestion. The significance
of bound phytochemicals to human health is not clear [27,
35]. However, it is possible that different plant foods with
different amounts of bound phytochemicals can be digested
and absorbed at different sites of the gastrointestinal tract
and play their unique health benefits. Bound phytochemi-
cals, mainly in β-glycosides, cannot be digested by human
enzymes and could survive stomach and small intestine di-
gestion to reach the colon and be digested by bacteria flora to
release phytochemicals locally to have health benefits [35].
0
20
40
60
80
100
120
0 0,08 0,17 0,25 0,33
Concentration of extract (mg/ml)
MDA production (% Control)
Free
Bound
0
20
40
60
80
100
120
0 0,08 0,17 0,25 0,33
Concentration of extract (mg/ml)
MDA production (% Control)
Free
Bound
a
b
Fig. 1 a Polyphenol extracts from Capsicum annuum var. aviculare
(Tepin) inhibit lipid peroxidation in Rat’s brain. bPolyphenol extracts
from Capsicum annuum var. aviculare (Tepin) inhibit lipid peroxidation
in Rat’s liver
Epidemiological studies have shown an inverse correlation
between vegetable consumption and colon cancer incidence
[36].
The protective ability of the free and bound phenolic ex-
tracts from Capsicum annuum var. aviculare (Tepin) is pre-
sented in Fig. 1a and b. Soluble free and bound phenolic ex-
tracts inhibited MDA production in cultured rat’s brain and
liver tissues in a dose-dependent manner. However, soluble
free phenols caused a significantly higher (P<0.05) inhi-
bition in the brain and liver tissues lipid peroxidation than
the bound phenolic compounds. The antioxidant activity of
phenolics is mainly because of their redox properties, which
allow them to act as reducing agents, hydrogen donors, free
radical scavenger, singlet oxygen quenchers and metal chela-
tors [37,38]. Furthermore, the EC50 of the soluble free and
bound phenolic extracts are presented in Table 2; the results
Table 2 EC50 of the red Capsicum annuum var. aviculare (Tepin)
extract
EC50 (mg/ml)
Sample Brain Liver
Free 0.11±0.02 b 0.06±0.01 b
Bound 0.19±0.03 a 0.15±0.02 a
Values represent means of triplicate.
Values with the same alphabet along the same column are not signifi-
cantly different (P>0.05).
revealed that the free soluble phenols [brain (0.11 mg/g),
liver (0.06 mg/g)] had a significantly higher EC50 than bound
phenolics [brain (0.19 mg/g), liver (0.15 mg/g)].
It is what noting that soluble free phenols and bound phe-
nols extract from this pepper caused a higher inhibition in the
MDA production in the brain of the rats than water-soluble
extracts of the same pepper [26]. This goes a long way to
confirm that, antioxidant activity of vegetables and fruits are
due mainly to their phenolics compounds [27], although one
cannot overrule the contribution from vitamin C and other
antioxidant phytochemicals. The basis for the difference in
the ability of the free and bound phenolic compounds to in-
hibit lipid peroxidation in both the brain and liver cannot
be categorically stated. However, it is very likely that β-
glycosides moiety in the phenolic compound in the bound
phenolic may have reduced the uptake of the bound pheno-
lics in the cultured brain and liver tissues, and this may have
reduce its antioxidant activity, unlike free soluble phenolics
that are readily absorbed in animal tissues [35]. Neverthe-
less, the protective ability of the bound phenolics against
lipid peroxidation may have contributed to the alleged pro-
tective ability of fruits and vegetables against colon cancer
[27,35].
However, incubation of the brain and the liver tissues in
the presence of 25 µM Fe(II) caused a significant increase
in the MDA content of both the brain and liver tissue. How-
ever, the percentage increase in the tissue MDA was higher
in the brain tissue (285%) than the liver tissues (238.2%).
The higher lipid peroxidation in the brain compared to the
liver could be attributed to the fact that brain cells, cannot
synthesis glutathione. Instead they rely on surrounding astro-
cyte cells to provide useable glutathione precursors, because
the brain has limited access to the bulk of antioxidants pro-
duced by the body, neurons are the first cells to be affected by
a shortage of antioxidants, and are most susceptible to ox-
idative stress. Researchers studying antioxidant protection
of neurons are finding short windows during development
of high vulnerability to oxidative stress [39]. High levels of
both Cu and Fe, with relatively low levels of Zn and Mn,
play crucial role in brain cancer and in degenerative diseases
of the brain (Parkinson’s and Alzheimer’s diseases, multiple
sclerosis, etc.) [5].
0
50
100
150
200
250
300
350
0 0,17 0,33 1,67 3,33
Concentration of extract (mg/ml)
MDA production (% Control)
Free
Bound
0
50
100
150
200
250
0 0,17 0,33 1,67 3,33
Concentration of extr act
(
m
g
/ml
)
MDA production (% Control)
Free
Bound
a
b
Fig. 2 a Polyphenol extracts from Capsicum annuum var. avicu-
lare (Tepin) inhibit Fe(II) induced lipid peroxidation in Rat’s brain.
bPolyphenol extracts from Capsicum annuum var. aviculare (Tepin)
inhibit Fe(II) induced lipid peroxidation in Rat’s liver
However, the free soluble and bound phenolic extracts
of the pepper caused a decrease in the MDA content of the
liver and brain in a dose dependent manner. Nevertheless,
a significant difference (P<0.05) in the ability of both free
and bound polyphenols to inhibit Fe(II)-induced lipid per-
oxidation in brain and liver was observed at higher dose
of the extract (1.67–3.33 mg/ml) not at lower dose (0.17–
0.33 mg/ml). However, the extract protected the liver than
the brain as shown in Fig. 2a and b. In addition, to the fact
that the inability of neurons to synthesis glutathione could
be responsible for the higher susceptibility of brain to lipid
peroxidation as earlier stated. The liver is very rich in antiox-
idant enzymes such as glutathione peroxidase, glutathione-
S-transferase, etc. as well as antioxidants metabolite such as
glutathione which is not readily available to brain cell, this
endogenous antioxidants may complemented the activity of
the exogenous antioxidants from the pepper. Furthermore,
soluble free phenols had higher protective effect than the
bound phenols. The basis for the difference in the protective
0
10
20
30
40
50
60
70
80
90
00,080,17 0, 25 0,33
Concentration of extract
(
m
g
/ml
)
%Fe (II) chelation
Free
Bound
Fig. 3 Fe(II) chelating ability of polyphenol extracts from Capsicum
annuum var. aviculare (Tepin)
effect of both polyphenol extracts cannot be categorically
stated, one possibility is that glucose of the bound polyphe-
nols may have reduce their antioxidant properties.
Antioxidant carries out their protective properties on cells
either by preventing the production of free radicals or by neu-
tralizing/scavenging free radicals produced in the body [40].
The mechanism through which the free and bound polyphe-
nols from the pepper protects liver and brain tissues from
Fe(II) induced lipid peroxidation was investigated, by deter-
mining the ability of the extract to chelate Fe(II) (preventing
production of free radicals) and the ability of the extracts to
scavenge OH radical. As shown in Fig. 3, both extracts had
high Fe(II) chelating ability. The result of the Fe(II) chelat-
ing ability was in agreement with the ability of the extract to
protect the brain and liver tissues from Fe(II) induced lipid
peroxidation, in that free polyphenols with the higher Fe(II)
chelating ability (Fig. 3) caused the highest inhibition in the
production of MDA in the brain and liver tissues (Fig. 2a and
b). Moreover, this clearly indicates that, one of the mecha-
nism through which pepper could protect the brain and liver
is through the ability of the polyphenols (free or bound) to
chelate Fe(II). Fe is usually accumulated in the brain, as one
grows older; and stored in the liver where they induced ox-
idative stress, which is largely responsible for both the brain
and liver damage.
Fe(II) induction of lipid peroxidation is through their par-
ticipation in Fenton reaction. The mechanism by which iron
can cause this deleterious effect is that Fe(II) can react with
hydrogen peroxide (H2O2) to produce the hydroxyl radical
(OH•) via the Fenton reaction, whereas superoxide can react
with iron(III) to regenerates iron(II) that can participate in
the Fenton reaction [41,42]. The overproduction of ROS
can directly attack the polyunsaturated fatty acids of the cell
membranes and induce lipid peroxidation [15]. As presented
in Fig. 4, both extracts significantly scavenge hydroxyl rad-
ical produced in Fe(II)/H2O2induced decomposition of de-
oxyribose in Fenton reaction in a dose-dependent manner.
Unlike our earlier study, where aqueous extracts of the same
pepper containing a mixture of vitamin C and polyphenols as
the main antioxidants could not significantly scavenge OH
radical [26]. However, free polyphenols from the pepper had
a higher OH radical scavenging ability in comparison with
the bound polyphenols. Nevertheless, OH radical scaveng-
ing ability of both the free and bound polyphenols from the
pepper (Fig. 4) are far below the Fe(II) chelating ability of
the extract (Fig. 3). This clearly shows that the domineering
mechanism through which Capsicum annuum var. avicu-
lare (Tepin) polyphenols protect the brain and the liver is
through their Fe(II) chelating ability rather than OH radical
scavenging. This gives credence, to the fact that the rec-
ommended therapy for Fe toxicity is through the use of Fe
chelator.
0
5
10
15
20
25
30
00,080,17 0,25 0,33
Concentration of extract (mg/ml)
OH radical scavenging ability (%)
Free
Bound
Fig. 4 OH radical scavenging
ability of polyphenol extracts
from Capsicum annuum var.
aviculare (Tepin)
0
50
100
150
200
250
300
00,81,6 2,4
Concentration of extract (mg/ml)
MDA Production (% Control)
Free
Bound
0
50
100
150
200
250
00,81,6 2,4
Concentration of extract (mg/ml)
MDA Production (% Control)
Free
Bound
a
b
Fig. 5 a Polyphenol extracts
from Capsicum annuum var.
aviculare (Tepin) inhibit sodium
nitroprusside induced lipid
peroxidation in Rat’s brain. b
Polyphenol extracts from
Capsicum annuum var.
aviculare (Tepin) inhibit sodium
nitroprusside induced lipid
peroxidation in Rat’s liver
Furthermore, NO radical has been reported to contribute
to degenerative diseases by reacting with superoxide radical
(O2∗) produced in Fenton reaction to form the powerful per-
oxynitrite (ONOO∗) which can caused damage to body cells.
Sodium nitroprusside, an anti-hypertensive drug, cause cy-
totoxicity through the release of cyanide and/or nitric oxide
(NO) [6]. As presented in Fig. 5a and b, incubation of the
brain and liver tissues in the presence of sodium nitroprusside
caused a significant increase (P<0.05) in MDA production
in the brain (247.7%) and liver (192.1%) tissues. However,
free and bound polyphenol extracts (0.8–1.24 mg/ml) from
the Capsicum annuum var. aviculare (Tepin) caused a signifi-
cant (P<0.05) decrease in the MDA content of the brain [free
(72.3–16.7%), bound (228.2–16.7%)] and liver [free (29.8–
17.3%), bound (142.0–16.7%)] in dose-dependent manner.
The inhibition of the sodium nitroprusside induced lipid
peroxidation in the liver and brain tissues, could be attributed
to the ability of the extract to scavenge the NO radical pro-
duced by the sodium nitroprusside, as well as chelating abil-
ity on the Fe produced from the decomposition of the sodium
nitroprusside. However, free polyphenols extract from the
pepper inhibited MDA production in the tissues than the
bound polyphenols; nevertheless, there was no significant
difference (P>0.05) in the inhibition of the lipid peroxida-
tion induced by sodium nitroprusside at higher concentration
of the extract, as shown in Fig. 5a and b. Therefore, NO radi-
cal scavenging ability of the polyphenols could contribute to
the protective ability of pepper against degenerative diseases.
Furthermore, incubation of the brain and liver tissues in
the presence of 1 mM quinolinic acid caused a significant
0
50
100
150
200
250
01,21,82,4
Concentration of extract (mg/ml)
MDA Production (% Control)
Free
Bound
0
50
100
150
200
250
01,21,82,4
Concentration of extract (mg/ml)
MDA Production (% Control)
Free
Bound
a
b
Fig. 6 a Polyphenol extracts
from Capsicum annuum var.
aviculare (Tepin) inhibit
quinolinic acid induced lipid
peroxidation in Rat’s brain. b
Polyphenol extracts from
Capsicum annuum var.
aviculare (Tepin) inhibit
quinolinic acid induced lipid
peroxidation in Rat’s liver
increase (P<0.05%) in the MDA content of the brain
(212.1%) and liver (187.9%) (Fig. 6a and b). Quinolinc
acid, a neuroactive metabolite of the tryptophan–kinurenine
pathway could caused oxidative stress in tissues by over-
stimulation of NMDA receptor and the production of free
radicals [7]. However, free and bound polyphenols from the
pepper caused a significant decrease (P<0.05) in the MDA
content of the brain and liver tissues in a dose-dependent
manner, except in liver tissues where the bound polyphenols
did not cause a significant decrease in the MDA content
in the liver (Fig. 5b). The mechanism through which
the polyphenols inhibited quinolinic acid induced lipid
peroxidation in the brain and liver could be, by inhibition of
over-stimulation of NMDA receptor by quinolinic acid.
Conclusion
In conclusion, Capsicum annuum var. aviculare (Tepin) con-
tains 83.7% free soluble polyphenol (218.2 mg/100 g) and
16.3% bound polyphenols (42.5 mg/100 g). In addition,
both polyphenolic extracts inhibit the various pro-oxidant
agents (Fe2+, sodium nitroprusside and quinolinic acid) in-
duced lipid peroxidation in brain and liver tissues in a dose-
dependent manner. However, the free polyphenols had higher
protective ability than the bound polyphenols. The main
mechanism through which they are carry out their protec-
tive effect against lipid peroxidation in the brain and the
liver is by Fe(II) chelating ability, OH and NO radicals scav-
enging ability and inhibition of over-stimulation of NMDA
receptor.
Acknowledgements The authors wish to acknowledge the Conselho
Nacional de Desenvolvimento Cient´
ıfico e Tecnol´
ogico (CNPq) Brazil
and Academy of Science for the Developing World (TWAS), Trieste
Italy; for granting Dr. G. Oboh, Post-Doctoral fellowship tenable at
Biochemical Toxicology Unit of the Department of Chemistry, Federal
University of Santa Maria, Brazil. This study was also supported by
CAPES, FIPE/UFSM, VITAE Foundation and FAPERGS. In addition,
the authors apppreciate the support of The Abdus Salam International
Centre for Theoretical Physics, Trieste, Italy. Financial support from the
Swedish International Development Cooperation Agency is acknowl-
edged.
References
1. Poli G, Albano E, Dianzani MU (1987) Chem Phys Lipids 45:117–
142
2. Sies H (1985) Oxidative stress, Academic Press, London
3. Hochstein P, Nordenbrand K, Ernster L (1964) Biochem Biophys
Res Commun 14:323–328
4. Benedetti A, Comporti M, Esterbauer H (1980) Biochim Biophys
Acta 620:281–296
5. Johnson S (2001) Med Hypo 57(5):539–543
6. Bates JN, Baker MT, Guerra R, Harrison DG (1990) Biochem
Pharm 42:S157–S165
7. Cabrera J, Reiter RJ, Tan D, Qi W, Sainz RM, Mayo JC, Garcia JJ,
Kim SJ, El-Sokkary G (2000) Neuropharm 39:507–514
8. Douglas CJ (1996) Trends Plant Sci 1:171–178
9. Harborne JB, Williams CA (2000) Phytochem 55:481–504
10. Pitchersky E, Gang DR (2000) Trends Plant Sci 5:459–445
11. Bors W, Heller W, Michel C, StettmaierK (1996) Handb Antioxid
409–466
12. Halliwell B (1996) Annu Rev Nutr 16:39–50
13. Rice-Evans C, Miller NJ, Paganga G (1996) Free Rad Biol Med
20:933–956
14. Rice-Evans CA, Miller J, Paganga G (1997) Trends Plant Sci
2:152–159
15. Elmegeed GA, Ahmed HA, Hussein JS (2005) Eur J Med Chem
40(12):1283–1294
16. Hollman PCH, Katan MB (1999) Food Chem Toxicol 37:937–942
17. Burda S, Oleszek W (2001) Food Chem 49:2774–2779
18. McGinnnis JM, Foege WHJ (1993) Amer Med Assoc 270:2207–
2212
19. Bronner YL (1996) J Am Diet Assoc 96:891–903
20. Munoz CM, Chavez A (1998) Int J Cancer 11(Ssuppl):85–89
21. Gillman MW, Cupples LA, Gagnon D, Posner BM, Ellison RC,
Castelli WP, Wolf PA (1995) J Am Med Assoc 273:1113–1117
22. Joshipura KJ, Ascherio A, Manson JE, Stampfer MJ, Rimm EB,
Speizer FE, Hennekens CH, Spiegelman D, Willett WC (1999) J
Am Med Assoc 282:1233–1239
23. Cox BD, Whichelow MJ, Prevost AT (2002) Public Health Nutr
3:19–29
24. Strandhagen E, Hansson PO, Bosaeus I, Isaksson B, Eriksson H
(2000) Eur J Clin Nutr 54:337–341
25. Sosulski F,Krygier K, Hogge L (1982) J Agric Food Chem 30:337–
340
26. Oboh G, Puntel RL, Rocha JBT (2005) Hot pepper (Capsicum
annuum, Tepin &Capsicum Chinese, Habanero)PreventsFe
2+-
induced Lipid Peroxidation in Brain – in vitro. A report submit-
ted to Conselho Nacional de Desenvolvimento Cient´
ıfico e Tec-
nol´
ogico (CNPq), Brazil
27. Chu Y, Sun J, Wu X, Liu RH (2002) J Agric Food Chem 50:6910–
6916
28. Belle NAV, Dalmolin GD, Fonini G, Rubim MA, Rocha JBT (2004)
Brain Res 1008:245–251
29. Ohkawa H, Ohishi N, Yagi K (1979) Anal Biochem 95:351–358
30. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Method
Enzym 299:152–178
31. Minotti G, Aust SD (1987) Free Rad Biol Med 3:379–387
32. Puntel RL, Nogueira CW, Rocha JBT (2005) Neurochem Res
30(2):225–235
33. Halliwell B, Gutteridge JMC (1981) FEBS Lett 128:347–352
34. Zar JH (1984) Biostatistical analysis, Prentice-Hall, Inc., USA
35. Sun J, Chu Y, Wu X, Liu R (2002) J Agric Food Chem 50:7449–
7454
36. Voorrips LE, Goldbohm RA, van Poppel G, Sturmans F, Hermus
RJJ, van den Brandt PA (2000) Am J Epidemiol 152:1081–1092
37. Amic D, Davidovic-Amic D, Beslo D, Trinajstic N (2003) Croat
Chem Act 76(1):55–61
38. Alia M, Horcajo C, Bravo L, Goya L (2003) Nutr Res 23:1251–
1267
39. Perry SW, Norman JP, Litzburg A, Gelbard HA (2004) J Neur Res
78(4):485–492
40. Oboh G (2006) Eur Food Res Technol (in press)
41. Harris ML, Shiller HJ, Reilly PM, Donowitz M, Grisham MB,
Bulkley GB (1992) Pharm Therap 53:375–408
42. Fraga CG, Oteiza PI (2002) Toxicology 80:23–32
