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Lactoferrin and ovotransferrin contribute toward antioxidative effects of Edible Bird’s Nest against hydrogen peroxide-induced oxidative stress in human SH-SY5Y cells


Abstract and Figures

There are reports of improved redox outcomes due to consumption of Edible Bird's Nest (EBN). Many of the functional effects of EBN can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of aging and its related diseases like Alzheimer's disease. In this study, the antioxidative potentials of EBN and its constituents, lactoferrin (LF) and ovotransferrin (OVF), were determined and protective effects against hydrogen peroxide (H2O2)- induced toxicity on SH-SY5Y cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange and propidium iodide (AO/PI) staining with microscopy were examined. Results showed that EBN and its constituents attenuated H2O2-induced cytotoxicity, and decreased radical oxygen species (ROS) through increased scavenging activity. Furthermore, LF, OVF, and EBN produced transcriptional changes in antioxidant related genes that tended towards neuroprotection as compared to H2O2-treated group. Overall, the results suggest that LF and OVF may produce synergistic or all-or-none antioxidative effects in EBN.
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Lactoferrin and ovotransferrin contribute toward antioxidative effects of
Edible Birds Nest against hydrogen peroxide-induced oxidative stress in
human SH-SY5Y cells
Zhiping Hou
, Mustapha Umar Imam
, Maznah Ismail
*, Nur Hanisah Azmi
Norsharina Ismail
, Aini Ideris
and Rozi Mahmud
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia;
Department of Nutrition and Dietetic, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
Serdang, Malaysia;
Department of Pathology, Chengde Medical University, Chengde, China;
Department of
Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia;
Department, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia
Received January 13, 2015; accepted April 21, 2015
There are reports of improved redox outcomes
due to consumption of Edible Birds Nest (EBN).
Many of the functional effects of EBN can be linked
to its high amounts of antioxidants. Interestingly,
dietary components with high antioxidants have
shown promise in the prevention of aging and its
related diseases like Alzheimers disease. In this
study, the antioxidative potentials of EBN and its
constituents, lactoferrin (LF) and ovotransferrin
(OVF), were determined and protective effects
against hydrogen peroxide (H
)- induced toxicity
on SH-SY5Y cells using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay and
acridine orange and propidium iodide (AO/PI)
staining with microscopy were examined. Results
showed that EBN and its constituents attenuated
-induced cytotoxicity, and decreased radical
oxygen species (ROS) through increased scavenging
activity. Furthermore, LF, OVF, and EBN produced
transcriptional changes in antioxidant related genes
that tended towards neuroprotection as compared to
-treated group. Overall, the results suggest that
LF and OVF may produce synergistic or all-or-none
antioxidative effects in EBN.
Key words: Edible Birds Nest; lactoferrin; ovotrans-
ferrin; antioxidant; neuroprotection
Aging is a slow and gradual biological process,
associated with multiple physiological and pathological
changes including altered redox status. The brain cells
are normally sensitive to the effects of reactive oxygen
species (ROS) because they are a nidus for peroxida-
tive molecules, and because of their peculiar energetic
In brain senescence, ROS starts to accumu-
late in neurons before clinically evident signs and
symptoms of the disease can be detected.
When ROS
accumulate, oxidative damage is normally prevented by
induction of protective factors like antioxidants, which
may not be effective if the insult is too overwhelming.
In such cases, apoptotic mechanisms set in to remove
neurons deemed irreparable.
Loss of neurons through
these apoptotic deaths results in severe morphological
and functional decits, which manifest with progressive
memory and cognitive decline.
Recently, due to concerns of side effects, researches
have been focused on natural substances with neuro-
protective potential that can scavenge free radicals and
protect cells from oxidative damage, rather than syn-
thetic chemicals.
Edible Birds Nest (EBN, Aerodra-
mus fuciphaga) is produced by swiftlets from their
salivary glue, which is a cementing substance.
Although EBN mainly contains carbohydrates, amino
acids, and mineral salts, its major ingredients are glyco-
Due to its nutritious and medical properties,
EBN has been deemed a precious food tonic in Chi-
nese community ever since Tang (608907AD) dynas-
Despite the long history of using EBN for
medicinal purposes, there has only been limited number
of scientic reports on the health benets of EBN.
Recently, EBN has been found to potentiate mitogenic
response of human peripheral blood monocytes
stimulate deoxyribonucleic acid synthesis in 3T3
Furthermore, enzymatic hydrolysis can
release active peptides, which have benecial effects on
a variety of biological systems
including the cardio-
vascular, gastrointestinal, immune, and nervous sys-
tems. The ability of glycoprotein to interact with
radical species or to inhibit oxidative reactions could
*Corresponding author. Email:
Abbreviations: AO/PI, Acridine orange and propidium iodide; EBN, Edible Birds Nest; LF, Lactoferrin; ORAC, Oxygen Radical Absorbance
Capacity; OVF, ovotransferrin; ROS, radical oxygen species; SOD, superoxide dismutase.
Bioscience, Biotechnology, and Biochemistry, 2015
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Downloaded by [Dr Imam Mustapha Umar] at 03:44 12 June 2015
lead to the development of novel food ingredients rele-
vant in health promotion and disease prevention.
Lactoferrin (LF) and ovotransferrin (OVF) are glyco-
proteins and family members of transferrin. Different
studies have also shown that LF and OVF serve neuro-
protective and antioxidant functions
and may there-
fore be good sources of functional food ingredients.
EBN is reported to have antioxidative properties,
and in view of the properties of LF and OVF, we
hypothesize that they may be contributing towards the
overall antioxidant properties of EBN.
SH-SY5Y cell line can be differentiated by retinoic
acid (RA) with similar characteristics to brain neurons
and thus can be used to study neuronal cell activity
in vitro.
Hydrogen peroxide (H
) is a major reac-
tive free radical that has been studied in a variety of
neurodegenerative diseases, and has been implicated as
an important mediator of unbalanced redox reactions
and apoptosis in various cells including neurons.
Consequently, this study was conducted to determine
the concentration of LF and OVF in EBN, and evaluate
their effectiveness against H
-induced oxidative
stress on SH-SY5Y cells.
Materials and methods
Materials. SH-SY5Y human neuroblastoma cell
line was obtained from American Type Culture Collec-
tion (Manassas, VA, USA). Minimum essential Eagles
medium, Hams nutrient mixture F-12 (DMEM/F-12),
fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO) and all other chemicals were purchased from
Sigma (St. Louis, MO, USA). The GenomeLabGeXP
Start Kit was from Beckman Coulter Inc. (Miami, FL,
USA), and the total ribonucleic acid (RNA) Isolation kit
was obtained from RBC Bioscience Corp. (Taipei, Tai-
wan). The OxiSelectSuperoxide Dimutase Activity
Assay (SOD) and OxiSelectIntracellular ROS Assay
kit (ROS) were from Cell Biolabs, Inc. (USA). Lactofer-
rin (L4894) and ovotransferrin (C7786) were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of EBN water-soluble protein. EBNs
from Sabah and Sarawak (states of Malaysia in the
Borneo Island) were dried in an oven at 50 °C for three
days and nely grounded with a food grinder (Waring
Commercial, Torrington, CT, USA). As reported previ-
the grounded EBN samples (1.0 g) were dis-
solved into 1000 ml of dd H
O, followed by
ultrasonication (Power Sonic 505, Hwashin Technology
Co. Seoul, Korea) in an ice bath (pulse on, 2 s; pulse
off, 4 s; ampl, 80%; 30 min) and centrifugation
(10,000 rpm for 10 min); the supernatants were
desalted and condensed in a dialysis bag with a 3500
cut-off molecular weight; then the water-soluble protein
stored at 20 °C until use.
LF and OVF detection. LF and OVF concentration
in EBN water extract were detected using Chicken
Lactoferrin Elisa kit and Chicken Ovotransferrin Elisa
Kit, Biosource (San Diego, CA, USA). All the proto-
cols were based on manufacturer instructions.
Cell culture. The human neuroblastoma SH-SY5Y
cells were grown in complete culture medium contain-
ing DMEM/F-12, 10% fetal bovine serum, 1% MEM
(non-essential amino acids), and 50 μg/ml gentamicin.
Cells were maintained at 37 °C with 5% CO
and 95%
2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) radical cation scavenging activity. ABTS
radical cation was generated through oxidation of
7 mM ABTS with 2.45 mM potassium persulfate and
incubated overnight in the dark at room temperature.
ABTS radical cation solution was then diluted with
ethanol to obtain an absorbance of 0.70 ± 0.02 at
734 nm spectrophotometerically (Pharmaspec UV-1700,
Shimadzu, Kyoto, Japan). EBN, LF and OVF (50 μL
each) were individually mixed with 950 μL of diluted
ABTS solution and incubated for 10 min at room tem-
perature. The absorbance was measured under 734 nm.
All the determinations were carried out in triplicate and
the readings were averaged. Trolox was used as stan-
dard and percentage of ABTS radical cation decoloriza-
tion inhibition was calculated using the formula
reported by Ismail in 2012.
ABTS radical cation scav-
enging activity of EBN, LF, and OVF was expressed in
mg Trolox equivalent per gram extracts as described
before (mg TE/g extract).
Oxygen Radical Absorbance Capacity (ORAC)
Assay. The abilities of extracts to scavenge peroxyl
radical capacity were determined by Oxygen Radical
Absorbance Capacity (ORAC) according to the method
described by Huang et al.
Briey, 150 μLuorescein
and 25 μL sample (1000 μg/ml EBN, 5 μg/ml LF, and
10 μg/ml OVF) or standard (Trolox) were added into
each well, and incubated 37 °C for 1015 min. After
incubation, uorescence measurements (Ex. 485 nm,
Em. 520 nm) were taken every one min to determine
the background signal. After 3 cycles, 25 μL of freshly
prepared 2,2-azobis(2-amidinopropane) dihydrochloride
(AAPH) at nal concentration 81.6 nM was added
manually with a multichannel pipette. Fluorescence
readings for each cycle were saved in Synergy H1
Hybrid Multi-Mode Microplate Reader (BioTek,
Winooski, VT, USA), as they would all be used
to calculate areas under the curves and Trolox
MTT assay. SH-SY5Y cells were seeded into
96-well culture plates at density of 2 × 10
cells/ml and
were allowed to attach. After 2 days, cells were differ-
entiated with RA (10 μM) for 7 days prior to treatment.
To examine the possible toxic effects, the cells were
treated with EBN (10100,000 μg/ml), LF (0.05
500 μg/ml), and OVF (0.11000 μg/ml) individually
for 24 h. To determine the neuroprotective ability, cells
were pretreated with 1000 μg/ml EBN and the equiva-
lence 5 μg/ml LF and 10 μg/ml OVF diluted in serum-
free medium for 24 h and then incubated with H
for another 2 h. Then, MTT was added to all the wells
and stranded in incubator for 4 h. The amount of MTT
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formazan product was dissolved by DMSO and mea-
sured absorbance at 540 nm using a Microplate reader
(All the MTT assays were performed in triplicate).
Acridine orange and propidium iodide (AO/PI)
staining. SH-SY5Y cells (2 × 10
) were seeded into
6-well culture plates, treated with 1000 μg/ml EBN and
the equivalence 5 μg/ml LF and 10 μg/ml OVF, then fol-
lowed by 250 μMH
for 2 h. Then, cells were har-
vested, and stained with AO/PI (10 μl of 1 mg/ml AO
and 10 μl of 1 mg/ml PI). They were examined under an
inverted uorescence microscope (Olympus, Tokyo,
Japan). Multiple independent images were taken.
Superoxide dismutase (SOD) and ROS ELISA
assays. SH-SY5Y cells (2 × 10
) were seeded into
12-well culture plates, treated with 1000 μg/ml EBN
and the equivalence 5 μg/ml LF and 10 μg/ml OVF,
then followed by 250 μMH
for 2 h. Protocols for
the ELISA tests were based on manufacturer instruc-
tions. For SOD assay, absorbance of samples were
nally read at 490 nm on the Synergy H1 Hybrid
Multi-Mode Microplate Reader (BioTek, Winooski, VT,
USA), while uorescence for ROS assay were read at
480/530 nm by Synergy H1 Hybrid Multi-Mode Micro-
plate Reader (BioTek, Winooski, VT, USA).
Ribonucleic acid (RNA) extraction, reverse transcrip-
tion and multiplex polymerase chain reaction (PCR)
analysis. SH-SY5Y-treated cells were extracted by
the Total RNA Isolation kit (RBC Bioscience Corp.,
Taiwan) according to the manufacturers instructions.
Primer sequences (Table 1) were designed using Geno-
meLab eXpress Proler software based on Homo
sapien sequence from the NCBI website. The primers
were supplied by Biosune (Shanghai, China), while the
internal control (KanR) was supplied by Beckman
Coulter. Reverse transcription and PCR were performed
according to the GenomeLabGeXP kit instruction
(Beckman Coulter) in an XP Thermal Cycler (Bioer
Technology, Germany). The PCR products were nally
analyzed by the GeXP genetic analysis system, and the
results were normalized using GeXpress Proler soft-
ware based on the manufacturers instructions.
Statistical analysis. All the data (n= 3) was con-
ducted by Tukeys multiple comparisons of one-way
ANOVA using Statistical Package for Social Science
(SPSS) version 20 (SPSS Inc., Chicago, IL). p<0.05
was considered as statistically signicant difference.
Results and discussion
LF and OVF detection in EBN water extraction
The results of LF and OVF concentrations deter-
mined using enzyme-linked immunosorbent assay
(ELISA) kits (chicken source), are shown in Table 2.
From comparisons made with the reported contents
from other sources, the compositions in EBN, espe-
cially white house EBN, are considerably higher. The
concentrations of LF and OVF in house white EBN
were approximately 0.5 and 1%, respectively. Thus, we
chose house white EBN as experimental subject.
Based on the biological functions of LF and OVF
, we hypothesized that LF and OVF could
be contributing towards the overall functional proper-
ties of EBN, which prompted further evaluation of their
EBN, LF, and OVF attenuate H
cytotoxicity on SH-SY5Y cells
The abilities of EBN, LF, and OVF to protect
SH-SY5Y cells from H
were determined by MTT
assay (Fig. 1), an indicator of cell viability. EBN
(10100,000 μg/ml), and equivalent concentrations
of LF an OVF in EBN (0.05500 μg/ml, and
0.11000 μg/ml, respectively) displayed above 80%
viability on SH-SY5Y cells (Fig. 1(A)), and showed no
signicant differences when compared with the control
group (p> 0.05).
is an oxidant that can induce intracellular
defense mechanisms to attenuate H
-induced oxida-
tive damage. As reported in our previous paper,
SH-SY5Y cells exposure to 250 μMH
for 2 h
resulted in approximately 50% cell death compared
with untreated control cells (p< 0.01). As shown in
Fig. 1(B), EBN produced a hormetic effect on the via-
bility of the cells in the presence of H
, with
1000 μg/ml EBN producing the best result. Although
antioxidants are known to relieve oxidative stress and
improve cell survival, it is also suggested that higher
concentrations of antioxidants may in fact disrupt the
natural hormetic capacity of cells
resulting in more
damage. It is also likely that at higher concentrations,
the oxidants simply became prooxidant.
for our subsequent experiments, 1000 μg/ml EBN, and
equivalent concentrations of LF and OVF (5 and
10 μg/ml, respectively) were used.
ABTS radical cation scavenging activity and ORAC
ORAC assay is a simple, sensitive, and reliable
method to quantitate the radical absorbing capacity of
antioxidants in serum or other biological uids,
especially natural products.
Additionally, ABTS is
also widely used for measuring the relative radical
scavenging activity of hydrogen donating and chain
breaking antioxidants in many elds.
To determine
antioxidant potentials of EBN, LF, and OVF, ABTS and
ORAC were performed on the samples (Fig. 2). EBN
(1000 μg/ml) exhibited the best scavenging effect on
ABTS (5.23 ± 0.18 mg Trolox equivalent/g extract),
followed by LF (5 μg/ml) and OVF (10 μg/ml) at con-
centrations similar to what is contained in 1000 μg/ml
of EBN (4.67 ± 0.40 and 2.79 ± 0.18 mg Trolox
equivalent/g extract, respectively). Results of the ORAC
assay also showed that EBN had the highest antioxidant
capacity (3.04 ± 0.23 mg Trolox equivalent/g extract)
when compared with LF and OVF (2.14 ± 0.10 and
1.22 ± 0.08 mg Trolox equivalent/g extract, respec-
tively). The results also indicated signicant differences
(p< 0.05) between LF and OVF, and also when each is
compared with EBN.
EBN lower oxidative stress 3
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Table 1. Gene name, accession number, reverse and forward primer sequences used in GeXP multiplex gene expression analyses.
Gene name Accession number
Primer sequence with universal tag
Forward Reverse
Internal control.
Housekeeping gene. RT reaction was at 48 °C for 1 min; 37 °C for 5 min; 42 °C for 60 min; 95 °C for 5 min, then hold at 4 °C, while PCR was as follows: initial denaturation at 95 °C for 10 min, followed by two-step cycles of 94 °C for 30 s and
55 °C for 30 s, ending in a single extension cycle of 68 °C for 1 min.
4 H. Zhiping et al.
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Results of EBN, LF, and OVF have demonstrated
consistent data in scavenging activities by ORAC and
ABTS radical scavenging assays. EBN demonstrated
the highest antioxidant activity followed by LF and
OVF via ORAC and ABTS radical cation scavenging
methods (Fig. 2). The results so far suggest that LF
and OVF possess antioxidant capacities, and even
though the capacities of any one of the compounds
may not explain the antioxidant capacity of EBN, it is
likely that they contribute towards the overall capacity
in EBN. Additionally, the overall antioxidant capacity
of EBN may be contributed by other compounds in
Table 2. Lactoferrin and Ovotransferrin expression in different species.
Lactoferrin Ovotransferrin
House white EBN (Sabah) 4.68 ± 0.57 μg/mg EBN 10.23 ± 0.72 μg/mg EBN
Cave white EBN (Sabah) 4.27 ± 0.49 μg/mg EBN 10.63 ± 0.90 μg/mg EBN
Cave black EBN (Sabah) 2.80 ± 0.21 μg/mg EBN 1.31 ± 0.07 μg/mg EBN
Cave red EBN (Sabah) 3.43 ± 0.07 μg/mg EBN 3.13 ± 0.58 μg/mg EBN
Cave red EBN (Sarawak) 3.13 ± 0.07 μg/mg EBN 8.40 ± 0.56 μg/mg EBN
Human milk 2 g/l
Cow milk 1 g/l
Human tears 0.9 g/l
Rat/rabbit/dog milk <50 mg/l
Hen egg NA 130 mg/g egg albumen
Notes: EBN, Edible Birds Nest; NA, not available.
Fig. 1. Effects of edible birdsnest (EBN), lactoferrin (LF), and ovotransferrin (OVF) on H
-induced cytotoxicity in SH-SY5Y cells deter-
mined by MTT assay.
Notes: (A) Human SH-SY5Y neuroblastoma cells were incubated with EBN (1010,000 μg/mL), LF (0.05500 μg/mL), and OVF (0.11000 μg/mL)
for 24 h; (B) SH-SY5Y pretreated with EBN, LF or OVF for 24 h with or without H
(250 μM) for an additional 2 h. Results are presented as the
mean ± SD in triplicates.
p< 0.01 vs. control.
EBN lower oxidative stress 5
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addition to LF and OVF, since LF and OVF do not
entirely reect the same activity as EBN. Different
studies have already shown that due to synergistic
effects of compounds in crude extracts, crude plant
extracts usually exhibit superior health-oriented proper-
ties than puried compounds.
It is also likely that
synergy between the different bioactive compounds in
EBN may potentiate their activity when they are bound
together in a single matrix.
AO/PI staining
AO and PI are among the most used stains for analy-
ses of cell apoptosis. Using a combination of these
stains, cells appear orange/red to indicate they are
apoptosis, while intact cells appear green. As shown in
Fig. 3, control cells appeared green, suggesting normal
structure and viability, while H
-treated cells
appeared orange, representing early apoptosis. As for
the EBN-treated cells, majority of the cells were stained
green, showing normal appearance of intact cells; simi-
larly, LF and OVF groups showed mostly normal cells,
with little signs of apoptosis.
ROS and SOD assay
SOD belongs to the endogenous antioxidant system
that can scavenge ROS. The disruption in the balance
between ROS and the endogenous antioxidant systems
contributes to oxidative damage of cellular macro-
molecules ultimately leading to cell death.
In this
study, the relative activity of SOD in the SH-SY5Y
cells exposed to 250 μMH
for 2 h decreased to
69.6% compared with the control group. Pretreatment
with EBN, LF, and OVF for 24 h recovered the SOD
activity to 90.7, 87.8, and 80.9%, respectively
(Fig. 4(A)). SOD is the important antioxidant defense
normally catalyze dismutation of the superoxide radical
into H
. However, SOD also displayed antioxidant
effect according to suppress the oxygen metabolites,
like superoxide.
To investigate whether EBN, LF, and OVF have the
effect to scavenge intracellular ROS generation induced
by H
, intracellular ROS kit was used to examine
intracellular H
/hydroxyl radical. DCFH-DA (2,7-
dichlorouorescin diacetate) uorescence staining indi-
cated that the value of H
-treated cells was increased
about twofold compared with untreated control cells.
However, the increase in intracellular H
reduced by EBN, LF, and OVF treatment (Fig. 4(B))
because they can directly attenuate hydroxyl radical
produced by H
. Meanwhile, the antioxidant system
will be stimulated even when exogenous ROS were
developed. As reported in the papers, OVF showed a
dose dependence in DCFH-DA oxidation by HD11
cells in the presence of phorbol 12-myristate 13-acetate
and LF displayed antioxidant ability in both
in ferric nitrilotriacetate-induced renal oxidative damage
in rats.
Therefore, the antioxidant properties of EBN,
LF, and OVF base on scavenging hydroxyl radical and
oxygen byproducts. Obviously, these results corroborate
those of the antioxidant capacities of LF and OVF,
which indicated that they had high antioxidant capaci-
ties that may be contributing to that of EBN.
Effects of EBN, LF, and OVF on mRNA levels of
antioxidant and apoptosis genes
The mRNA levels of three antioxidant and apoptosis
genes (poly (ADP-ribose) polymerase 1 [PARP1],
SOD1, and SOD2, nuclear factor kappa-light-chain-en-
hancer of activated B cells [NF-κB], cellular tumor
antigen p53 [p53], p38 mitogen-activated protein kinase
[p38MAPK], and protein kinase B [Akt]) were studied
using Multiplex GeXP genetic analysis system, with
KanR as the internal control. The target genes and
housekeeping gene are shown in Table 1.
As shown in Fig. 5, treatment with 250 μMH
downregulated the mRNA levels of SOD1, SOD2, and
PARP1 genes. Treatments with 1000 μg/ml EBN water
extract, 5 μg/ml LF, or 10 μg/ml OVF upregulated the
expression of the three genes even in the presence of
250 μMH
. In the case of SOD1 and SOD2, the
EBN, LF, and OVF-treated cells upregulated the gene
signicantly higher than H
-treated controls
(p< 0.01), although the expression levels were lower
than in non-treated control cells (p< 0.01). The mRNA
level of PARP1 in EBN treated cells was higher than
-treated and normal untreated cells (p< 0.05), but
those of LF- and OVF-treated cells were not different
from H
-treated cells, and were lower than in
untreated controls (p> 005).
Upregulation of antioxidant genes (SOD) is an
endogenous mechanism for boosting antioxidant
defenses in the presence of oxidative insults, and could
be the basis for the increased SOD activity observed in
this study when SH-SY5Y cell were treated with EBN,
LF, or OVF (Fig. 4). Moreover, the mRNA levels for
the EBN, LF, and OVF groups are reective of the
SOD activities observed in the respective groups, as no
signicant differences in SOD mRNA levels or activi-
ties were observed between the groups. There are
numerous papers reported the antioxidant ability of
lactoferrin, most importantly, ovotransferrin was also
processed SOD-like function
which showed the main
Fig. 2. ABTS radical cation scavenging activities and ORAC of
Edible Birds Nest, lactoferrin (LF), and ovotransferrin (OVF).
Notes: Concentrations of LF and OVF used for the assays corre-
spond to their concentrations present in EBN, as shown in Table 2.
Values expressed as mean ± standard deviation.
p< 0.01 vs. EBN;
p< 0.01 vs. LF. ABTS, 2,2-azino-bis[3-ethylbenzothiazoline-6-sul-
fonic acid; ORAC, oxygen radical absorbance capacity.
6 H. Zhiping et al.
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key enzyme involved in redox balance in cellular and
controlled antioxidant expression both in proteomic and
transcriptional levels. The results, therefore, suggest
that EBN boosts SOD levels and its activity through
increased transcriptional regulation of the gene, and
that LF and OVF in EBN contribute to this effect.
However, combination of these compounds and others
in EBN may not have produced additive effects since
there are no differences between EBN, LF, and OVF
treatments. Furthermore, activation of PARP1 is
reported to improve cell survival after oxidative dam-
age from H
Its increased expression by EBN but
not LF or OVF suggest that other constituents in EBN
may be contributing to this effect other than LF or
OVF, or that their synergistic effect with or without
other compounds may be contributing to expression
Fig. 3. AO (acridine orange, green)/PI (propidium iodide, red) double staining on SH-SY5Y human cells under uorescent microscope.
Notes: (A) untreated control cells; (B) treatment with 250 μMH
for 2 h; (CE) cells pretreated with 1000 µg/ml EBN water extract, 5 µg/mL
LF or 10 µg/mL OVF, respectively, and subsequently treated with 250 μMH
for 2 h.
Fig. 4. Superoxide dismutase (SOD) activity (A) and reactive oxygen species (ROS) generation (B) in SH-SY5Y cells, following treatment with
1000 μg/ml EBN water extract, 5 µg/mL LF, or 10 µg/mL OVF, and subsequent treatment with or without 250 μMH
, in comparison with
untreated control. Results are presented as the mean ± SD in triplicates.
p< 0.01 vs. H
p< 0.01 vs. control,
p< 0.01 vs. EBN.
EBN lower oxidative stress 7
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level seen in EBN-treated cells. The overall data from
this study, therefore, indicates that EBN constituents
interact in different ways to produce its activity; the
presence of multiple compounds may produce additive
or synergistic effects as suggested by the antioxidant
capacity tests and expression of PARP1, while in some
cases it may produce an all-or-none effect as seen with
the activity and expression of SOD.
In summary, the present study demonstrated that
EBN protects SH-SY5Y cells against H
cytotoxicity and cell oxidative stress. LF and OVF con-
tributed to the antioxidative effects of EBN in different
ways. They were also able to inhibit early apoptosis in
response to H
treatment. The ndings indicate that
EBN constituents contribute towards producing syner-
gistic antioxidative effects of EBN.
Author contributions
Study design: Hou Zhiping, Maznah Ismail. Supervi-
sion of the study: Maznah Ismail, Aini Idris and Rozi
Mahmud. Primer design for gene expression study:
Norsharina Ismail and Mustapha Umar Imam. Conduct
of experimental parts: Hou Zhiping and Nur Hanisah
Azmi. Data analyses and preparation of manuscript:
Mstapha Umar Imam and Hou Zhiping. Review of
manuscript and nal approval for submission: Maznah
This work was carried out under the nancial sup-
port of Ministry of Science, Technology and Innova-
tion, e-sciencefund (vote 5450666), Malaysia. The
authors wish to thank Mr. Mohd Khairil Othman from
Matrix Optics (M) Sdn. Bhd. for the technical assis-
tance in uorescence microscopy analysis and all staff
of Laboratory of Molecular Biomedicine for the help
during the study.
Disclosure statement
The authors declare that they have no competing interests.
Mustapha Umar Imam
Nur Hanisah Azmi
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, in comparison to untreated control and treatment with 250 μMH
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EBN lower oxidative stress 9
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... An in vitro study showed that pre-treatment with EBN could reduce intracellular ROS [24]. The experimental model used was SH-SY5Y human neuroblastoma cells stimulated with hydrogen peroxide (H 2 O 2 ). ...
... SH-SY5Y cells stimulated with H 2 O 2 experience oxidative stress due to the increase in hydroxyl radicals and subsequently undergo a cascade of apoptotic consequences [26]. The possible pathway in reducing ROS proposed by Hou et al. [24] is via free radical scavenging activity of EBN and increased SOD activity through increased expression of SOD genes. However, SOD is responsible for the breakdown of highly reactive O 2 into less reactive H 2 O 2 and oxygen and has no direct effect on H 2 O 2 [18]. ...
... These features include shrinkage, chromosome condensation, DNA fragmentation, and the presence of apoptotic bodies. Both Yew et al. [27] and Hou et al. [24] showed that EBN could inhibit apoptosis. It was found that there were reduced morphological changes when neurotoxic cells were pre-treated with EBN. ...
Neurodegenerative diseases are often associated with the accumulation of oxidative stress and neuroinflammation. Edible bird’s nest (EBN) is a glycoprotein (sialylated mucin glycopeptides) found to be beneficial against neurodegenerative diseases. Antioxidative, anti-inflammatory, and anti-apoptotic properties of EBN in preserving neuronal cells were widely researched using in vitro and in vivo models. Functional effects of EBN are often linked to its great number of antioxidants and anti-inflammatory glycopeptides. Bioactive compounds in EBN, especially sialic acid, add value to neurotrophic potential of EBN and contribute to neuronal repair and protection. Various studies reporting the neuroprotective effects of EBN, their molecular mechanisms, and neuroactive composition were gathered in this review to provide better insights on the neuroprotective effects of EBN.
... Edible bird's nests antioxidant properties are attributed to the inclusion of several bioactive components, including amino acids, sialic acid, triacylglycerol, vitamins, lactoferrin, fatty acids, minerals, and glucosamine (70,72,73). Due to the inclusion of two key components, ovotransferrin and lactoferrin, EBN displayed antioxidative action (74). In addition, the researchers proved their capacity to protect human neuroblastoma SH-SY5Y (HNS) cells against the toxicity caused by hydrogen peroxide (H 2 O 2 ). ...
... Thus, EBN may be a viable nutraceutical option for preventing neurodegenerative diseases related to oxidative stress. In a second study, Hou et al. (74) revealed the impact of EBN on the toxicity depletion of H 2 O 2 in HNS cells. Lactoferrin and ovotransferrin were reported to protect against H 2 O 2 -induced toxicity and cytotoxicity when incorporated in EBN. ...
Full-text available
Cognitive enhancement is defined as the augmentation of the mind's core capabilities through the improvement of internal or external information processing systems. Recently, the focus has shifted to the potential therapeutic effects of natural products in improving cognitive function. Edible bird's nest (EBN) is a natural food substance derived from the saliva of swiftlets. Until today, EBN is regarded as a high-priced nutritious food with therapeutic effects. The effectiveness of dietary EBN supplementation to enhance brain development in mammals has been documented. Although the neuroprotection of EBN has been previously reported, however, the impact of EBN on learning and memory control and its potential as a cognitive enhancer drug remains unknown. Thus, this article aims to address the neuroprotective benefits of EBN and its potential effect as a cognitive enhancer. Notably, the current challenges and the future study direction in EBN have been demonstrated.
... Moreover, 50 µg/mL of the peptides had a weaker protective effect on PC12 cells from SNP-induced injury, while with the concentration up to 400 µg/mL, the cell viability increased to 85.66 ± 1.40%, which was similar to the effect of 100 µM edaravone (positive control). Our finding suggested that the purified peptides (P2) could reduce SNP-induced PC12 cell damage and enhance cell viability, which agreed with the effect of EBN extract to attenuate H 2 O 2 -induced oxidative damage on human neuroblastoma cells [47]. μg/mL) for 2 h, cell viability was prominently boosted with a dose-effect (p < 0.05). ...
... Our finding suggested that the purified peptides (P2) could reduce SNP-induced PC12 cell damage and enhance cell viability, which agreed with the effect of EBN extract to attenuate H2O2-induced oxidative damage on human neuroblastoma cells [47]. ...
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In this research, the neutrase hydrolysis conditions of edible bird’s nest (EBN) by-products were optimized by response surface methodology (RSM). Antioxidant peptides were then isolated from the EBN by-products by ultrafiltration and chromatography taking the DPPH radical scavenging ability as an indicator. The antioxidant activity of the purified peptides was estimated by radical scavenging ability and sodium nitroprusside (SNP)-induced damage model in PC12 cells. When the enzyme concentration was10 kU/g-hydrolysis temperature was 45 °C, and hydrolysis time was 10.30 h, the degree of hydrolysis (DH) of EBN by-product hydrolysate (EBNH) was the highest. The purified peptide exerted strong scavenging ability with EC50 values of 0.51, 1.31, and 0.65 mg/mL for DDPH, ABTS, and O2− radicals, respectively. In addition, the purified peptides could significantly reduce the SNP-induced oxidative damage of PC12 cells, and twelve peptides that were rich in leucine (Leu), valine (Val), and lysine (Lys) were identified by LC-MS/MS. These results suggested that EBN by-products have potential as new materials for natural antioxidant peptides.
... The pellet cells were then suspended in 10 g/mL 1:1 AO/PI mixture. Cell morphological alteration were then examined using a fluorescent microscope CoolSNAP-Pro digital camera (Olympus) (Al-Harbi et al., 2020;Hou et al., 2015). ...
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Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxi-dant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chi-nese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apo-ptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover , the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore , to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes. K E Y W O R D S apoptosis, nimbin, polycystic ovarian syndrome, testosterone, zebrafish
... EBN is also being studied as an anti-aging agent to maintain good health and slow down the aging process and diseases (Chua et al. 2013;Hou et al. 2015;Yew et al. 2019). According to Careena et al. (2018), EBN significantly improves memory and confirms neuroprotective activity against neuroinflammatory inhibition and oxidative stress processes. ...
Full-text available
Edible bird's nest (EBN) is a salivary secretion of swiftlets which consist of protein and carbohydrate rich glycoproteins. This natural ingredient is very valuable, nutritional and medically valuable. The EBN industry have grown rapidly and benefited the Malaysian economy, hence, it is viewed seriously and it is actively supported by the government. This review discusses the progress and development of EBN industry as well as the R&D activities and endeavours especially that which involves deriving peptides with biological activities from EBN and its by-product sources. Many studies have documented the therapeutic properties of EBN such as antiaging, antiviral, antioxidant, and antihypertensive. Studies have also been conducted to produce glycoprotein hydrolysates from EBN through enzymatic hydrolysis, and findings showed that these bioactive peptides increase solubility as well as antioxidant and antihypertensive activities. Enzymatic hydrolysis breaks long protein chains at specific sites and releases amino acids and small peptides with lower molecular weights. The EBN hydrolysates produced can improve bioactivity and overcome insolubility and low absorption of EBN prepared and consumed through traditional means. Further studies need to be carried out to optimise EBN glycoprotein hydrolysates production as well as maximising their bioavailability and efficacy in the human gastrointestinal system. In addition, EBN by-products produced during EBN cleaning process should be fully utilised to recover the high-value glycoproteins, while reducing pollution and wastage. By enhancing R&D activities of EBN, bioactive glycopeptides produced from EBN may become an important functional food ingredient for various uses and innovative value-added products in the future.
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Edible bird's nests (EBNs), a food of animal origin, are temporary nests built by swiftlets to foster their offspring. As EBNs and their products are widely accepted by consumers, the safety of hormones in EBNs has also received increasing attention. The establishment of a method for hormone analysis in EBNs and the investigation of hormone levels based on the analytical method are the most effective measures to eliminate any safety concerns. In this study, a multi-residue method was developed for the simultaneous determination of 45 hormones in EBNs, including estrogens, progesterones, androgens, and cortical hormones. EBN samples (1.0 g) were weighed into 50 mL polypropylene centrifuge tubes and mixed with 8 mL of pure water. Then, the samples were extracted twice with 15 mL of acetonitrile and ethyl acetate (1∶1, v/v) under ultrasonic-assisted conditions for 30 min, and the protein in the EBN samples was precipitated at 4000 r/min for 5 min. The clear supernatants were loaded onto a hydrophilic-lipophilic balanced (HLB) SPE column, which was previously activated with methanol (3 mL) followed by pure water (3 mL). The cartridge was washed with 3 mL of pure water and 3 mL of 50% methanol aqueous solution. The hormones were eluted with 3 mL of methanol. A rapid analysis was performed using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The hormones in the extracting solution were separated on a Phenomenex Kinetex C18 column (100 mm×2.1 mm, 2.6 μm) and eluted by gradient elution with acetonitrile-0.1% formic acid aqueous solution (ESI+) or acetonitrile-water (ESI-). Qualitative and quantitative analyses were performed using the multiple reaction monitoring (MRM) mode. The HPLC-MS/MS results showed good linearity in the range of 0.20-20.0 μg/L with correlation coefficients (R2) ≥0.9990. For the 45 hormones, the limits of detection (LODs, S/N≥3) were 0.04-0.70 μg/kg and the limits of quantification (LOQs, S/N≥10) were 0.16-2.00 μg/kg. The recoveries of five hormones, namely, fluorometholone, budesonide, aldosterone, fluocinonide, and ethinylestradiol, were 40.2%-63.6%. Owing to the low recoveries, this method might be suitable only for qualitative testing of the five hormones. The recoveries of the other 40 target analytes were between 72.2% and 112.3% at spiked levels of 2.0, 4.0, and 20.0 μg/kg with relative standard deviations (RSDs) of 2.5%-11.6%. The method is characterized by easy operation, rapidness, high precision, and high sensitivity for the 40 compounds. Thus, this method is suitable for determination of the 40 hormones from EBNs for qualitative testing and quantitation. The proposed method was used to analyze 1021 EBN samples from Malaysia, Indonesia, Thailand, and Vietnam from 2017 to 2021. Only three hormones, progesterone, boldenone, and androstenedione, were identified in the EBN samples, while the levels of the other hormones were lower than their individual LODs. The detected rates of progesterone, boldenone, and androstenedione were 100%, 79%, and 89%, respectively. The contents of progesterone, boldenone, and androstenedione in the EBN samples were 0.097-3.58 μg/kg, 0-0.096 μg/kg and 0-1.77 μg/kg, respectively. The levels of hormones in EBNs were compared with those in eggs, pure milk, and dairy products, which were also animal-derived foods. Androstenedione was detected in all egg samples monitored, and its content was higher than that in EBN samples, pure milk, and dairy products. The content of boldenone was similar among the four products investigated in this study. Based on risk assessment using progesterone, the dietary intake was found to be 3.54 μg/d from milk >1.09 μg/d from eggs >0.0030 μg/d from EBNs. The results showed that the levels of hormones in EBNs were much lower than those in eggs, milk, and dairy products for daily consumption. Based on this investigation, the health effect of the hormones in EBNs may be insignificant.
This study aimed to investigate the immunomodulatory effect of edible bird’s nest (EBN) in vitro and in vivo studies. EBN-treated peripheral blood mononuclear cells showed that EBN specifically enhanced the expansion of CD3⁺ T-cells. The restoration of lymphocyte subpopulations under the influence of immunosuppressive drug has been successfully recovered in CD3⁺ T-cells, not CD45RA⁺ B-cells and CD335⁺ NK-cells. In addition, oral administration of EBNs in Sprague-Dawley rats revealed their potential to increase the number of peripheral blood T-cells. Our study also demonstrated that EBN treatments affected the numbers of the Peyer’s patches, spleen weight and length, and cellularity of the periarteriolar lymphoid sheath. Interestingly, we observed that elevation of serum interleukin-2 (IL-2) had been correlated with the proliferation of T-cells in the animal model. Therefore, these results are essential for developing therapeutic strategies in improving immunity, particularly T-cell homeostasis, under immunosuppressive therapy.
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Diluents and various biological products have been used in different animal species, with promising outcomes in post-thaw sperm quality. Nevertheless, only a few reports are available for the semen of Arabian horses. Edible bird’s nest (EBN) – a product of the salivary secretions of swiftlet species is widely known to have both antioxidant and anti-inflammatory properties. Presently, there is no data available on the role of EBN supplemented in different extenders and its effect on semen quality in stallion semen. Two in vitro experiments were conducted to examine the effects of edible bird’s nest (EBN) on the quality of chilled and post-thawed cryopreserved Arabian stallion spermatozoa. In experiment one, 10 ejaculates were collected, divided into two equal parts, diluted using EquiPlus® and INRA 96® and supplemented with 0 % (control), 0.12 %, 0.24 % EBN concentrations. The semen samples were stored at 5 ℃ and observed at 0, 24, and 48 h. Sperm kinetics variables (% total motility [TM] and progressive motility [PM], curvilinear velocity; VCL, straightness; VSL, average path velocity; VAP) were analyzed using computerized assisted sperm analysis. For chilled semen, there was no significant difference in any of the sperm quality parameters between control (0 %), 0.12 %, and 0.24 % EBN supplementation either in INRA96® or EquiPlus®. In experiment two, nine ejaculates were diluted and cryopreserved using EquiPlus Freeze® and INRA Freeze® containing 0 %, 2.4 %, and 4.8 % EBN, and evaluated after thawing. Sperm kinetics, DNA integrity and antioxidant capacity - Biological Anti-oxidant Potential (BAP) and Reactive Oxygen Metabolites (d-ROMs) test were evaluated. In chilled semen, there was no significant difference in any of the sperm quality parameters between control (0 %), 0.12 %, and 0.24 % EBN supplementation either in INRA96® or EquiPlus®. For frozen semen supplemented with 2.4 % and 4.8 % EBN had higher sperm motility parameters compared to control in INRA Freeze® and EquiPlus Freeze®, but the values were not statistically significant (P > 0.05). Also, EBN supplementation had no significant effects on the DNA integrity, biological antioxidant potential, and reactive oxygen metabolites. EBN supplementation had no significant effects on sperm quality and antioxidant status in chilled and frozen Arabian Stallion semen. Future studies might consider different methods of EBN preparation and concentrations to elucidate the potential biological impact of EBN in Arabian stallion semen
Edible bird's nest (EBN) swiftlet existed naturally 48,000 years ago in caves as their natural dwellings. Nowadays, edible bird's nest has become a very important industry due to its high nutritional, medicinal and economic value. Additionally, edible bird's nest has a long quality guarantee period. Obviously, the nutritional components and medicinal functions vary depending on geographical origins. Recently, the global demand for edible bird's nest has markedly increased, accompanied by the increasing attention of all key players of the global food trade system, i.e., producers, consumers, traders and the authorities to obtain safe and high-quality edible bird's nest. Hence, this target can be accomplished via the enforcement of an efficient and universal geo-tracing technique. Current methods of the geo-tracking of edible bird's nest, i.e., automation, physical and analytical techniques have several limitations and all of them fail to discriminate different quality grades of edible bird's nest. Meanwhile, in many studies and applications, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) has proven to be a “cutting edge” technique for greatly enhance food traceability from field to fork through its ability in distinguishing the food products in terms of their quality and safety. This article provides an overview of (1) edible bird's nest as a multiuse strategic food product, (2) quality issues associated with edible bird’s nest including implications that the site of acquisition of the edible bird’s nest has food safety implications, (3) current regulations and geo-tracking approaches to ensure the safety and quality of edible bird’s nest with the special focus on polymerase chain reaction-denaturing gradient gel electrophoresis technique as a vigorous and universal geo-tracing tool to be suggested for edible bird's nest geo-traceability.
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Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting the senile population with manifestation of motor disability and cognitive impairment. Reactive oxygen species (ROS) is implicated in the progression of oxidative stress-related apoptosis and cell death of the midbrain dopaminergic neurons. Its interplay with mitochondrial functionality constitutes an important aspect of neuronal survival in the perspective of PD. Edible bird's nest (EBN) is an animal-derived natural food product made of saliva secreted by swiftlets from the Aerodamus genus. It contains bioactive compounds which might confer neuroprotective effects to the neurons. Hence this study aims to investigate the neuroprotective effect of EBN extracts in the neurotoxin-induced in vitro PD model. Methods: EBN was first prepared into pancreatin-digested crude extract and water extract. In vitro PD model was generated by exposing SH-SY5Y cells to neurotoxin 6-hydroxydopamine (6-OHDA). Cytotoxicity of the extracts on SH-SY5Y cells was tested using MTT assay. Then, microscopic morphological and nuclear examination, cell viability test and ROS assay were performed to assess the protective effect of EBN extracts against 6-OHDA-induced cellular injury. Apoptotic event was later analysed with Annexin V-propidium iodide flow cytometry. To understand whether the mechanism underlying the neuroprotective effect of EBN was mediated via mitochondrial or caspase-dependent pathway, mitochondrial membrane potential (MMP) measurement and caspase-3 quantification were carried out. Results: Cytotoxicity results showed that crude EBN extract did not cause SH-SY5Y cell death at concentrations up to 75 μg/ml while the maximum non-toxic dose (MNTD) of water extract was double of that of crude extract. Morphological observation and nuclear staining suggested that EBN treatment reduced the level of 6-OHDA-induced apoptotic changes in SH-SY5Y cells. MTT study further confirmed that cell viability was better improved with crude EBN extract. However, water extract exhibited higher efficacy in ameliorating ROS build up, early apoptotic membrane phosphatidylserine externalization as well as inhibition of caspase-3 cleavage. None of the EBN treatment had any effect on MMP. Conclusions: Current findings suggest that EBN extracts might confer neuroprotective effect against 6-OHDA-induced degeneration of dopaminergic neurons, particularly through inhibition of apoptosis. Thus EBN may be a viable nutraceutical option to protect against oxidative stress-related neurodegenerative disorders such as PD.
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Previous studies on postmortem human brain tissue have shown that the iron-binding glycoprotein lactoferrin is up-regulated in dopamine (DA) neurons resistant to degeneration in Parkinson disease (PD). To study how this could possibly relate to disease progression, we used midbrain cultures and experimental settings that model the progressive loss of DA neurons in this disorder. Human lactoferrin of either recombinant or natural origin provided robust protection to vulnerable DA neurons in a culture paradigm where these neurons die spontaneously and selectively as they mature. The efficacy of lactoferrin was comparable to that of GDNF, a prototypical neurotrophic factor for DA neurons. Neuroprotection by lactoferrin was attributable to its binding to heparan sulfate proteoglycans onto the cell surface of DA neurons and subsequently to partial inactivation of focal adhesion kinase (FAK), a major effector kinase of integrins. We established that FAK inactivation served to unmask a prosurvival phosphoinositide 3-kinase (PI3K)/AKT-dependent signaling pathway that stimulates calcium shuttling from endoplasmic reticulum to mitochondria. DA neurons exposed to the mitochondrial toxin 1-methyl-4-phenylpyridinium were also partially protected by lactoferrin, comforting the view that mitochondria may represent a downstream target for lactoferrin protective actions. Finally, we found that the iron binding capability of lactoferrin intervened in DA cell rescue, only when neurodegeneration was consecutive to iron-catalyzed oxidative stress. Overall, our data suggest that the accumulation of lactoferrin in PD brains might be evidence of an attempt by the brain to minimize the consequences of neurodegeneration.
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Oxidative stress is implicated in the pathogenesis of diabetic complications, and can be increased by diet like white rice (WR). Though brown rice (BR) and germinated brown rice (GBR) have high antioxidant potentials as a result of their bioactive compounds, reports of their effects on oxidative stress-related conditions such as type 2 diabetes are lacking. We hypothesized therefore that if BR and GBR were to improve antioxidant status, they would be better for rice consuming populations instead of the commonly consumed WR that is known to promote oxidative stress. This will then provide further reasons why less consumption of WR should be encouraged. We studied the effects of GBR on antioxidant status in type 2 diabetic rats, induced using a high-fat diet and streptozotocin injection, and also evaluated the effects of WR, BR and GBR on catalase and superoxide dismutase genes. As dietary components, BR and GBR improved glycemia and kidney hydroxyl radical scavenging activities, and prevented the deterioration of total antioxidant status in type 2 diabetic rats. Similarly, GBR preserved liver enzymes, as well as serum creatinine. There seem to be evidence that upregulation of superoxide dismutase gene may likely be an underlying mechanism for antioxidant effects of BR and GBR. Our results provide insight into the effects of different rice types on antioxidant status in type 2 diabetes. The results also suggest that WR consumption, contrary to BR and GBR, may worsen antioxidant status that may lead to more damage by free radicals. From the data so far, the antioxidant effects of BR and GBR are worth studying further especially on a long term to determine their effects on development of oxidative stress-related problems, which WR consumption predisposes to.
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The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H 2 O 2) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H 2 O 2 -mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.
Free radicals are generated in the brain during normal intake of oxygen, during infection, and during normal oxidative metabolism of certain substrates. Oxidative damage is highly relevant in the brain because it is exposed to high oxygen concentrations-utilizing about one-fifth of the oxygen consumed by the body; contains relatively poor concentrations of antioxidants and related enzymes; is enriched in iron, which is a potent catalyst for oxidative species formation; and is rich in polyunsaturated fatty acids, which are prone to oxidation. It is widely accepted that OS increases during aging, and it can be considered an important age-dependent factor making the brain more susceptible to several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis. This chapter focuses primarily on OS associated with disturbances in energy metabolism, with special emphasis on the role of mitochondria and redox metal ions as the principal sources of OS. The last part of the chapter discusses of the role of AD lesions, senile plaques, and neurofibrillary tangles as potential neuronal compensatory responses against OS.
Neurodegenerative disorders affect almost 30 million individuals leading to disability and death. These disorders are characterized by pathological changes in disease-specific areas of the brain and degeneration of distinct neuron subsets. Despite the differences in clinical manifestations and neuronal vulnerability, the pathological processes appear similar, suggesting common neurodegenerative pathways. Apoptosis seems to play a key role in the progression of several neurologic disorders like Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis as demonstrated by studies on animal models and cell lines. On the other hand, research on human brains reported contradictory results. However, many dying neurons have been detected in autoptic brains of patients with neurodegenerative diseases, and these conditions are often associated with significant cell loss accompanied by typical morphological features of apoptosis such as chromatin condensation, DNA fragmentation, and activation of cysteine-proteases called caspases. Cell death and neurodegenerative conditions have been linked to oxidative stress and imbalance between generation of free radicals and antioxidant defenses. Multiple sclerosis, stroke, and neurodegenerative diseases have been associated with reactive oxygen species and nitric oxide. Here we present an overview of the involvement of neuronal apoptosis and oxidative stress in the most important neurodegenerative diseases, mainly focusing the attention on several genetic disorders, discussing the interaction between primary genetic abnormalities and the apoptotic pathways.
Understanding and treating vernal keratoconjunctivitis (VKC) has been a challenge because the pathogenesis is unclear and antiallergic therapy often unsuccessful. The aim of the study was to analyze peptide profiles in human tears using mass spectrometry to elucidate compositional differences between healthy subjects and patients affected by VKC. Tears were collected from healthy subjects and VKC patients. Digested samples were treated with iTRAQ (isobaric tag for relative and absolute quantitation). Separation of tryptic peptides was realized using a MicroHPLC interfaced with a microfraction collector. MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer. Protein Pilot™ software with Paragon™ algorithm v4.1.46 or GPS™ with Mascot engine was used as search engines with SwissProt or IPI human as the databases. A significant number of peptides were examined, and 78 proteins were successfully identified. In all VKC samples, levels of serum albumin, transferrin, and hemopexin were found up to 100 times higher than control tear levels and correlated to the severity of disease. Hemopexin, transferrin, mammaglobin B, and secretoglobin 1D were found significantly over-expressed in VKC samples compared with the control samples. Tear samples from patients treated with topical cyclosporine or corticosteroids showed a dramatic reduction in these protein levels. LC MALDI MS and isobaric tag for relative and absolute quantitation technique may be useful in the quantitative and qualitative characterization of the peptidoma of human tears. These techniques may identify target proteins to be used in the diagnosis and management of VKC and other inflammatory ocular surface conditions.
To better understand the molecular mechanisms underlying the anti-aging properties of water extract of edible bird’s nest (WEBN), the effect of WEBN on oxidative stress-induced matrix metalloproteinase-1 (MMP-1) was assessed in human HaCaT keratinocyte. Upon exposure to H2O2, WEBN inhibited H2O2-induced cytotoxicity and scavenged intracellular reactive oxygen species, down-regulated H2O2-induced MMP-1 mRNA expression, protein expression and activity, and lowered the activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinases (JNK), which are the upstream of MMP-1 expression. Furthermore, WEBN inhibited the expression of c-Fos and phospho c-Jun, which are components of the transcription factor activator protein-1 (AP-1), AP-1 transcriptional activity, and AP-1 binding to the MMP-1 promoter. These data indicate that the anti-aging properties of WEBN involve the inhibition of MMP-1 expression via down-regulation of the ERK /JNK and AP-1 pathway.
Milk provides a well-balanced source of amino acids and other ingredients. One of the functional ingredients in milk is lactoferrin (LF). LF presents a wide variety of bioactivities and functions as a radical scavenger in models using iron-ascorbate complexes and asbestos. Human clinical trials of oral LF administration for the prevention of colon polyps have been successful and demonstrated that dietary compounds exhibit direct interactions. However, antioxidative properties of LF in distant organs require further investigation. To study the antioxidant property of LF, we employed bovine lactoferrin (bLF) using the rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal tubular oxidative injury. We fed rats with bLF (0.05%, w/w) in basal chow for 4 weeks and sacrificed them after Fe-NTA treatment. After intraperitoneal administration of 9.0 mg iron/kg Fe-NTA for 4 and 24 h, bLF pretreatment suppressed elevation of serum creatinine and blood urea nitrogen levels. In addition, we observed protective effects against renal oxidative tubular damage and maintenance of antioxidant enzyme activities in the bLF-pretreated group. We thus demonstrated the antioxidative effect of bLF against Fe-NTA-induced renal oxidative injury. These results suggest that LF intake is useful for the prevention of renal tubular oxidative damage mediated by iron.
The neuroprotective effect of Nigella sativa oil (NSO) and its fractions against beta amyloid (Aβ)-induced cell death in primary rat cerebellar granule neurons was investigated. Primary cultures were pretreated for 5 h with NSO and its fractions – hexane fraction (HF), ethyl acetate fraction (EAF) and water fraction (WF) – before incubating with 10 µM Aβ1-40 for 24 h. Cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and lactate dehydrogenase (LDH) assays. Results of the MTS assay showed that the WF and NSO were significantly protective against cell death induced by 10 µM Aβ1-40 at 1, 10 and 100 µg/mL probably because of antioxidant properties as determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical trapping method. HF and EAF had low DPPH scavenging effect and were only effective at 100 µg/mL. However, NSO and its fractions were weakly protective against cell membrane damage in the LDH assay. NSO and its fractions, especially the WF, may play a role in the prevention of Aβ-induced cell death. Neurodegeneration in Alzheimer's disease (AD) has been associated with the toxic effects of beta amyloid (Aβ), a by-product of amyloid precursor protein. Natural products that are able to reduce Aβ-induced neurotoxicity are candidates for preventing as well as for treating this neurodegenerative condition. Thus, the neuroprotective effects of Nigella sativa against Aβ toxicity may play a potential role in preventing AD progression.