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Antioxidant activities and phenolics content of eight species
of seaweeds from north Borneo
Patricia Matanjun & Suhaila Mohamed &
Noordin Mohamed Mustapha & Kharidah Muhammad &
Cheng Hwee Ming
Received: 1 March 2007 /Revised and Accepted: 10 August 2007 / Published online: 2 May 2008
# Springer Science + Business Media B.V. 2007
Abstract The antioxidant activity of eight edible species of
Malaysian North Borneo seaweeds obtained from Sabah
waters (Kudat, Tanjung Aru and Semporna) consisting of
three red seaweeds (Eucheuma cottonii, E. spinosum and
Halymenia durvillaei), two green seaweeds (Caulerpa
lentillifera and C. racemosa) and three brown seaweeds
(Dictyota dichotoma, Sargassum polycystum and Padina
sp.) were determined. Methanol and diethyl ether were used
as extraction solvent. The antioxidant activities were
determined by two methods, TEAC (trolox equivalent
antioxidant capacity) and FRAP (ferric reducing antioxidant power) assays. The total phenolic content of the
extract was determined according to the Folin–Ciocalteu
method and results were expressed as phloroglucinol
equivalents. The methanolic extracts of green seaweeds,
C. lentillifera and C. racemosa, and the brown seaweed, S.
polycystum showed better radical-scavenging and reducing
power ability, and higher phenolic content than the other
seaweeds. The TEAC and FRAP assays showed positive
and significantly high correlation (R
2
=0.89). There was a
strong correlation (R
2
=0.96) between the reducing power
and the total phenolic content of the seaweeds methanolic
dry extracts. These seaweeds could be potential rich sources
of natural antioxidants.
Keywords Seaweeds
. Antioxidant activity . TEAC . FRAP.
Phenolics
. North Borneo waters
Introduction
Reactive oxygen species (ROS) and oxidative stress have
been associated with the onset of a variety of chronic
disease states in humans, including coronary heart disease
(CHD), certain cancers, rheumatoid arthritis, diabetes,
retinopathy of prematurity, chronic inflammatory disease
of the gastrointestinal tract, as well as diseases associated
with cartilage, Alzeimer’s disease (Chauhan and Chauhan
2006), other neurological disorders and the ageing process
(Temple 2000). ROS are toxic as they can oxidize
biomolecules leading to cell death and tissue injury
(Morrisey and O’Brien 1998). Conversely, antioxidants
are believed to be protective because they may help to
protect the human body against damage by ROS (Halliwell
et al. 1995). Antioxidants from natural sources are preferred
by consumers (Kranl et al. 2005) due to concerns on the
toxic and carcinogenic effects of synthetic antioxidants (Ito
et al. 1986; Safer and Al-Nughamish 1999).
J Appl Phycol (2008) 20:367–373
DOI 10.1007/s10811-007-9264-6
P. Matanjun
School of Food Science and Nutrition, Universiti Malaysia Sabah,
Locked Bag 2073,
88999 Kota Kinabalu, Sabah, Malaysia
S. Mohamed (*)
: K. Muhammad
Faculty of Food Science & Technology,
Universiti Putra Malaysia,
UPM 43400 Serdang,
Selangor, Malaysia
e-mail: mohamed.suhaila@gmail.com
N. M. Mustapha
Faculty of Veterinary Medicine, Universiti Putra Malaysia,
UPM 43400 Serdang,
Selangor, Malaysia
C. H. Ming
Department of Physiology, Faculty of Medicine,
Universiti Malaya,
50603 Kuala Lumpur, MalaysiaMarine algae are exposed to a combination of light and
oxygen that leads to the formation of free radicals and other
strong oxidizing agents (Dykens et al. 1992; Namiki 1990),
but the absence of oxidative damage in the structural
components (polyunsaturated fatty acids) of seaweeds
(Matsukawa et al. 1997) and their stability to oxidation
during storage (Ramarathnam et al. 1995) suggest their cells
have protective antioxidative defense systems (JiménezEscrig et al. 2001). Marine algae contain phloroglucinol
phenolics (phlorotannins) (Pavia and Aberg 1996) which are
probably good antioxidants (Ragan and Glombitza 1986),
since plant phenolics can behave as ROS scavengers, metal
chelators and enzyme modulators and prevent lipid peroxidation (Rodrigo and Bosco 2006). Polyphenols are reducing
agents, and together with other dietary reducing agents such
as vitamin C, E and carotenoids, referred to as antioxidants,
protect the body’s tissues against oxidative stress and
associated pathologies such as cancer, coronary heart disease
and inflammation (Tapiero et al. 2002).
The antioxidant activity of several seaweeds has been
published (Matsukawa et al. 1997; Anggadiredja et al.
1997; Jiménez-Escrig et al. 2001; Yan et al. 1998; Rupérez
et al. 2002; Nagai and Yukimoto 2003), but there are no
publications on the antioxidant activities of seaweeds from
Malaysian North Borneo (Sabah). Since marine algae are
rich source of dietary fiber, minerals, proteins and vitamins
(Yan et al. 1998), a documented antioxidant activity of
these seaweeds would elevate their value in the human diet
as food and pharmaceutical supplements.
Many authors have stressed the need to carry out more
than one type of antioxidant activity measurement to take
into account the various mechanisms of antioxidant action
(Frankel and Meyer 2000; Prior and Cao 1999), as no
single assay will accurately reflect all of the radical sources
or all antioxidants in a mixed or complex system (Prior et
al. 2005). In the present study, we evaluated the antioxidant
activities of seaweeds using trolox equivalent antioxidant
capacity (TEAC) and ferric reducing antioxidant power
(FRAP) assays. These two analytical methods are routinely
used to assess antioxidant activities in vitro. We also
estimated the total phenolic contents of these seaweeds
using the classical Folin–Ciocalteu reagent, and investigated the relationship between the total antioxidant capacities
and phenolic contents in the seaweed samples.
Materials and methods
ABTS (2,2 -azino-bis-(3ethylbenzothiozoline-6-sulfonic acid)′
diammonium salt), TPTZ (2,4,6-tri(2-pyridyl)-S-triazine),
potassium persulfate (K2S2O8), Folin-Ciocalteu reagent,
butylated hydroxytoluene (BHT) and quercetin were purchased from Sigma (USA). Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), a water analogue of
vitamin E, was purchased from Aldrich (USA). Phloroglucinol (trihydroxybenzen) was obtained from Fluka Chemicals (Switzerland) and Ferric chloride hexahydrate
(FeCl3
.6H2O) came from BDH (England). Sodium sulphate
anhydrous, methanol, diethyl ether, and hydrochloric acid
were from Merck (Germany). All other reagents were of
analytical grade.
Specimens of the eight seaweed species were collected
from the coastal areas of Sabah, Malaysia. Eucheuma cottonii
and E. spinosum were harvested from the Universiti
Malaysia Sabah farms in Bangi, Kudat (north coast of
Sabah). Caulerpa lentillifera and C. racemosa were collected from Semporna (east coast of Sabah) and Tanjung Aru
(west coast of Sabah) waters, respectively. Halymenia
durvillaei, Dictyota dichotoma, Sargassum polycystum and
Padina spp. were collected from Tanjung Aru waters (west
coast of Sabah). Fresh seaweeds were washed with distilled
water and their holdfasts and epiphytes removed. The fresh
seaweed was placed in a freezer (−20°C) immediately after
collection. All cleaned seaweeds were freeze-dried at −50°C
for 3 days and then ground to fine powder using a Waring
miller to pass through a 0.5-mm screen, and stored in airtight containers at −20°C.
Two extraction procedures were carried out according to
the modified method of Mohd Zin et al. (2002) and
Vairappan (2003). The procedures were as follows. First,
ground freeze-dried seaweed samples (10 g) were extracted
with 100 mL of methanol at room temperature for 72 h. The
samples were then filtered with Whatman filter paper no. 1
and the solvent removed under vacuum at 40°C using a
rotary evaporator to give a dark green viscous mass. This
extract is the methanolic dry extract. Second, ground
freeze-dried seaweed samples (10 g) were extracted with
100 mL of methanol at room temperature for 72 h. Samples
were filtered and the methanol solution was concentrated in
vacuo and partitioned between diethyl ether and water
(ratio 1:3). Diethyl ether was washed with water, dried over
anhydrous sodium sulphate and evaporated to leave a dark
green oil. This extract is the diethyl ether dry extract.
The methanol extract was used since it has shown to
give the highest antioxidant activity in many seaweed
species (Yan et al. 1999). Previous study had shown that, in
the case of methanol extraction, the content of polyphenols
at 72 h was higher than at 24 h (unpublished data),
therefore extraction was conducted for 72 h to maximize
the yield of polyphenols. According to Lapornik et al.
(2005), yield of polyphenols in alcohol extracts strongly
increases with a longer time of extraction. The dried
extracts were weighed and the yield extracts obtained were
calculated. The solvent extract that gave higher yield was
further subjected to TEAC and FRAP assays for comparison of their antioxidant activities.
368 J Appl Phycol (2008) 20:367–373TEAC assay
The seaweed extracts (1 mg dry extract diluted in 1 mL
methanol) antioxidant activities were measured using the
TEAC method as described by Re et al. (1999) with some
modifications. The reaction was carried out in a microtiter
plate. Briefly, ABTS
+
radical cation was generated by a
reaction of 7 mM ABTS with 2.45 mM potassium
persulfate. The reaction mixture was allowed to stand in
the dark for 16 h at room temperature. Working solution
was prepared by diluting 1 mL of ABTS stock solution
with 19 mL phosphate buffered saline (PBS, 5 mM, pH 7.4)
where 200 μL of this working solution was dispensed to
each well of microtitration plate. Addition of 20 μL diluted
methanolic extracts (20 μL of sample extract was diluted
with 60 μL of methanol) initiated the reaction and
absorbance was read after exactly 6 min. A microplate
reader was used to read the absorbance at 645 nm. Trolox, a
vitamin E analogue in the concentration of 0 to 2.5 mM,
was used as standard and calibration. BHT and quercetin
whose concentrations were 1 mg mL
−1
, respectively, were
used as positive controls. All measurements were performed in triplicate. The results are expressed as mM
Trolox.mg
−1
dry weight of extract.
FRAP assay
The seaweed extracts (1 mg dry extract diluted in 1 mL
methanol) antioxidant activities were measured using FRAP
assay according to the Benzie and Strain (1999) method with
some modifications. The reaction was carried out in a
microtiter plate. The antioxidant activity of the standards was
estimated by using the increase in absorbance caused by the
generated ferrous ion. The working FRAP reagent contained
300 mM acetate buffer (pH3.6), 10 mM TPTZ 40 mM HCl
and 20 mM FeCl3.6H2O in the ratio of 10:1:1, freshly
prepared and warmed to 37°C. Two hundred μL of this
working solution was dispensed to each well of the microtitration plate. Then, addition of 20 μL diluted methanol
extracts (20 μL of sample extract was diluted with 60 μL of
methanol) initiated the reaction and absorbance was read
after exactly 10 min. A microplate reader was used to read
the absorbance at 593 nm. Trolox, in the concentration of 0
to 1000 μM, was used as standard and for calibration. BHT
and quercetin whose concentrations were 1 mg/1mL respectively were used as positive controls. All measurements were
performed in triplicate. The results were expressed as μM
Trolox.mg
−1
dry weight of extract.
Total phenolic content
The total phenolic (TP) content of the extract was determined
according to the Folin–Ciocalteu method (Velioglu et al.
1998) using phloroglucinol (a basic structural unit of
phlorotannins) as a standard and expressing results as
phloroglucinol equivalents (PGE) (Jiménez-Escrig et al.
2001). A 1.0 mL-aliquot of sample was added to 1.5 mL of
deionized water and 0.5 mL of Folin–Ciocalteu reagent (10×
dilution), and the contents were mixed thoroughly. After 1
min, 1.0 mL of 20% sodium carbonate solution was added,
and the mixture was again mixed thoroughly. The controls
contain all the reaction reagents except the sample. After
30 min kept in the dark, the absorbance was measured at
750 nm, and compared to a phloroglucinol calibration curve.
Statistical analysis
All data are expressed as means ± standard deviation. Data
were analyzed using one-way analysis of variance (ANOVA)
followed by Duncan multiple range tests by using the SPSS
system version 11.5 for Windows. Pearson’s correlation test
was used to assess correlations between means. A significant
difference was considered at the level of p<0.05.
Results
Table 1 shows the extraction yields of the methanolic and
diethyl ether extracts on a dry weight basis. The methanolic
extracts showed higher yield as compared with the diethyl
ether extracts for all seaweed samples. The extraction yield
of the methanolic dry extracts ranged from 1.88% to
40.33% while the diethyl ether dry extracts ranged from
0.47% to 10.87%.
Table 2 shows the results of primary screening of
antioxidant activity of all methanolic extracts using TEAC
assay expressed as TEAC value. This value represented
mM Trolox equivalents mg
−1
dry extract. The antioxidant
activities of the seaweed samples ranged from 1.49±0.02 to
2.16±0.04 mM Trolox equivalents mg
−1
dry extract. The
sequence of antioxidant activity of the methanolic seaweed
extract determined by TEAC assay was as follows: C.
Table 1 Extraction yield of seaweeds methanolic and diethyl ether
extracts on dry weight basis
Seaweeds Division Yield (%) w/w
Methanol Diethyl ether
Eucheuma cottonii Rhodophyta 2.25 0.86
Eucheuma spinosum Rhodophyta 1.88 0.59
Halymenia durvillaei Rhodophyta 7.92 1.68
Caulerpa lentillifera Chlorophyta 30.86 5.74
Caulerpa racemosa Chlorophyta 26.70 7.36
Dictyota dichotoma Phaeophyta 40.33 10.87
Sargassum polycystum Phaeophyta 4.05 0.47
Padina spp. Phaeophyta 8.55 2.87
J Appl Phycol (2008) 20:367–373 369lentillifera > C. racemosa > S. polycystum > D. dichotoma >
H. durvillaei > E. cottonii > E. spinosum > Padina spp.
The reducing activity of the seaweed extracts as
determined by FRAP assay varied as seen in Table 2. The
sequence of antioxidant activity of the methanolic seaweed
extract determined by FRAP assay was as follows: C.
lentillifera > C. racemosa > D. dichotoma > Padina spp. >
S. polycystum > E. cottonii > H. durvillaei > E. spinosum.
Table 3 shows the brown and green seaweeds have higher
phenolic content compared with the red seaweeds. The
sequence of total phenolic content of the methanolic seaweed
extracts was as follows: S. polycystum > C. lentillifera > C.
racemosa > D. dichotoma > Padina spp. > E. cottonii > H.
durvillaei > E. spinosum.
Figure 1 shows a relationship between free radicalscavenging activity, as TEAC value, and the reducing
power, as FRAP value, of eight species of seaweed crude
methanolic extracts. The correlation gave R
2
=0.89 indicating strong correlation between TEAC and FRAP assays.
Figure 2 shows a relationship between FRAP and TP values
which gave a correlation of R
2
=0.96 indicating strong
correlation. Figure 3 shows a relationship between TEAC
and TP which gave a correlation of R
2
=0.56 indicating
good correlation but not as strong as the correlation
between FRAP and TP values.
Discussion
The results presented in Table 1 show that there was an
increase in yield with increased solvent polarity (polarity
index of methanol and diethyl ether are 5.1 and 2.8,
respectively), an indication that more polar compounds were
found in seaweed extracts. Variation in the yields of various
extracts is attributed to polarities of different compounds
present in the plants. Since methanolic extracts gave significantly higher yield than diethyl ether for all seaweeds, the
methanolic extracts (dry weight basis) were used and tested
for antioxidant activity using TEAC and FRAP assays.
There are many methods to determine antioxidant
capacity. These methods differ in terms of their assay
principles and experimental conditions; consequently, in
different methods antioxidants in particular have varying
contributions to total antioxidant potential (Cao and Prior
1998). In this study, the seaweed extracts antioxidant
activities were tested using two different assays, TEAC
and FRAP assays. These two methods represented different
mechanisms of antioxidant action. A sample possessing
TEAC free radical-scavenging activity indicated that its
mechanism of action was as a hydrogen donor and
terminated the oxidation process by converting free radicals
to more stable products, whereas a compound exhibiting a
positive result in the FRAP assay was an electron donor and
it terminated the oxidation chain reaction by reducing the
oxidized intermediates into the stable form (Tachakittirungrod
et al. 2007).
Table 3 Total phenolic (TP) contents of seaweeds methanolic dry
extracts expressed as phloroglucinol equivalents (PGE)
Seaweeds TP mg PGE /g dry extract
Eucheuma cottonii 22.50±2.78
d
Eucheuma spinosum 15.82±1.24
e
Halymenia durvillaei 18.90±1.03
de
Caulerpa lentillifera 42.85±1.22
ab
Caulerpa racemosa 40.36±1.05
b
Dictyota dichotoma 35.23±5.65
c
Sargassum polycystum 45.16±3.01
a
Padina spp. 33.11±3.96
c
Values are expressed as mean±standard deviation, n=3
Different superscript letters within a column indicate significant
differences between samples at the level of p<0.05
10
9
4
7
5
8
2
3
1
6
0.0
1.0
2.0
3.0
4.0
5.0
0.0 100.0 200.0 300.0 400.0 500.0 600.0 700.0
FRAP values (uM/mg)
TEAC values (mM/mg)
Fig. 1 Correlation of FRAP and TEAC values of seaweed methanolic
dry extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)
Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dictyota dichotoma, (7) Sargassum polycystum, (8) Padina
spp, (9) BHT and (10) Quercetin
Table 2 Antioxidant activities of seaweeds methanolic dry extracts
determined by TEAC and FRAP assays
Seaweeds TEAC
(mM.mg
−1
dry
extract)
FRAP
(μM.mg
−1
dry
extract)
Eucheuma cottonii 1.63±0.09
fg
225.00±11.31
e
Eucheuma spinosum 1.54±0.08
gh
153.97±8.07
f
Halymenia durvillaei 1.67±0.04
e
182.29±13.35
f
Caulerpa lentillifera 2.16±0.04
c
362.11±15.65
c
Caulerpa racemosa 2.01±0.04
d
355.36±20.65
c
Dictyota dichotoma 1.66±0.09
f
268.86±13.30
d
Sargassum polycystum 1.86±0.02
h
366.69±11.85
c
Padina spp. 1.49±0.07
h
251.43±14.07
de
Butylated
hydroxytoluene
3.84±0.06
a
615.71±29.73
a
Quercetin 3.65±0.08
b
557.36±19.99
b
Values are expressed as mean±sandard deviation, n=3
Different superscript letters within a column indicate significant
differences between samples at the level of p<0.05
370 J Appl Phycol (2008) 20:367–373The TEAC assay applied in this study was according to
the improved technique described by Re et al. (1999) for the
generation of ABTS
•-
which involves the direct production
of the blue/green ABTS
•-
chromophore though the reaction
between ABTS and K2S2O8. This method is applicable to the
study of both water-soluble and lipid-soluble antioxidants,
pure compounds and food extracts. According to Ragan and
Glombitza (1986), radical-scavenging capacity of seaweeds
methanol extracts might be mostly related to their phenolic
hydroxyl group. In the present study, the green seaweeds
Caulerpa lentillifera and C. racemosa had greater antioxidant activities compared to the brown and red seaweeds. The
positive controls, BHT and quercetin, showed extremely
high antioxidant activity (TEAC values were above 3.0)
while all the seaweed samples showed high antioxidant
activity (TEAC values were below 3.0 but above 1.0). None
of the seaweed samples showed moderate antioxidant
activity (TEAC values below 1.0 but above 0.5) and low
antioxidant activity (TEAC values below 0.5).
In the FRAP assay, antioxidants in the sample reduce Fe
3+
/
tripyridyltriazine complex (Fe
3+
-TPTZ), present in stoichiometric excess, to the blue-colored ferrous form (Fe
2+
-TPTZ).
The antioxidant potential is propotional to the combined
(total) ferric reducing/antioxidant power (FRAP value) of the
antioxidants in the sample (Benzie and Szeto 1999). The reducing power property indicates that the antioxidant compounds are electron donors and can reduce the oxidized
intermediates of the lipid peroxidation process, so that they
can act as primary and secondary antioxidants (Yen and Chen
1995). We found that the green seaweeds C. lentillifera and
C. racemosa and the brown seaweed Sargassum polycystum
were more reactive then the other seaweeds, which were
similar to the results obtained in the TEAC assay. The
positive controls, BHT and quercetin, showed significantly
higher antioxidant activity than all the seaweed samples.
In this study, strong positive correlation between TEAC
and FRAP assays (R
2
=0.89) indicated the compounds
present in the methanolic extracts of seaweeds capable of
reducing ABTS radical were also able to reduce ferric ions.
Pulido et al. (2000) reported, in general, that the ferric ion
reducing ability of antioxidant correlates with the results
from other methods used to estimate antioxidant capacity.
Similarly, Thaipong et al. (2006) also reported high
correlation between TEAC and FRAP assays.
The phenolic contents of these seaweeds were evaluated
using the Folin–Ciocalteu method. The variation of phenolic
content was quite large (Table 3). The brown seaweed S.
polycystum and green seaweed C. lentillifera showed
significantly higher phenolic content than all the red
seaweeds. Jiménez-Escrig et al. (2001) also reported similar
findings that brown seaweeds contained higher phenolic
content than the red seaweeds. In agreement with previous
studies (Nagai and Yukimoto 2003; Duan et al. 2006), there
was a significant correlation between antioxidant activity and
phenolic content of these eight species of seaweeds. Many
algal species contain polyphloroglucinol phenolics (phlorotannins) (Pavia and Aberg 1996; Nakamura et al. 1996) and
in this study the antioxidant activity of algae could be due to
these compounds. The phenolic content in the seaweed
extracts showed a much higher correlation with reducing
power (R
2
=0.96) than the radical-scavenging activity (R
2
=
0.56). The lower correlation between TEAC values and the
phenolic contents in the seaweed extracts indicated that not
only the phenolic compounds were involved in the antioxidant activity through this pathway but there might be some
effects involving other active compounds.
Conclusions
Methanol showed higher extraction yield than diethyl ether
indicating more polar compounds were found in these
2
3
1
8
5
4
7
6
0.0
10.0
20.0
30.0
40.0
50.0
0.0 0.5 1.0 1.5 2.0 2.5
TEAC values (mM/mg dry extract)
TP (mg PGE/g dry extract)
Fig. 3 Correlation of TEAC and TP content of seaweed methanolic
extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)
Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dicyota dichotoma, (7) Sargassum polycystum and (8)
Padina spp
7
4
5
6
8
1
3
2
0.0
10.0
20.0
30.0
40.0
50.0
0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0
FRAP values (uM/mg dry extract)
TP (mg PGE/g dry extract)
Fig. 2 Correlation of FRAP and TP content of seaweed methanolic
extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)
Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dictyota dichotoma, (7) Sargassum polycystum and (8)
Padina spp
J Appl Phycol (2008) 20:367–373 371seaweed extracts. The green seaweeds C. lentillifera and C.
racemosa and the brown seaweed S. polycystum showed
better radical-scavenging and reducing power ability, and
higher phenolic content, than the other seaweeds. These
seaweeds could be potential rich sources of natural antioxidants. The TEAC and FRAP assays showed positive and
significantly high correlation (R
2
=0.89). There was a strong
correlation (R
2
=0.96) between the reducing power and the
total phenolic content of the seaweedsmethanolic extracts
expressed as phloroglucinol equivalents. The present findings appear useful in leading to further study in the
identification and characterization of specific compounds
responsible for the relatively high antioxidant activities in
these seaweeds. These studies are now in progress.
Acknowledgements The authors would like to thank Borneo
Marine Research Institute at the Universiti Malaysia Sabah and Sabah
Fisheries Department for supplying the seaweeds, Dr. Charles
Vairappan for collection of seaweeds and technical assistance, and
Teh Ooi Kock of University Malaya for their technical assistance in
the antioxidant assays. This study was funded by the Ministry of
Science, Technology and Innovation of Malaysia.
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