Phytochemical Profile, Antioxidant and Cytotoxic Activities of the Carob Tree (Ceratonia siliqua L.) Germ Flour Extracts

Abstract

This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.

Full text

ORIGINAL PAPER
Phytochemical Profile, Antioxidant and Cytotox ic Activiti es
of the Carob Tree (Ceratonia siliqua L.) Germ Flour Extracts
Luísa Custódio & Ana Luísa Escapa & Eliana Fernandes & Alba Fajardo & Rosa Aligué &
Fernando Alberício & Nuno Neng & José Manuel Florêncio Nogueira & Anabela Romano
Published online: 12 March 2011
#
Springer Science+Business Media, LLC 2011
Abstract This work aimed to evaluate the phytochemical
content and to determine the antioxidant and cytotoxic
activities of methanol extracts of the carob tree (Ceratonia
siliqua L.) germ flour. The extracts were rich in phenolic
compounds, had considerable antioxidant activity, and
reduced the viability of cervical (HeLa) cancer cells. The
chemical content and the biological activities of the extracts
were significantly affected by gender and cultivar. Female
cultivar Galhosa had the highest levels of phenolic com-
pounds, and the highest antioxidant activity. Extracts from
the hermaphrodite trees and from the female cultivars
Galhosa and Costela/Canela exhibited the highest cytotoxic
activity. The most abundant compound was theophylline.
The phenolic content was correlated to both antioxidant and
cytotoxic activities. Our findings provide new knowledge
about the health implications of consuming food supple-
mented with carob germ flour.
Keywords Alkaloids
.
Antioxidant
.
Antiproliferative
.
Oxidative stress
.
Phenolic compounds
.
ROS
.
Theophylline
Abbreviations
RSA Radical scavenging activity
Introduction
The carob tree (Ceratonia siliqua L.) is one of the most
useful trees of the Mediterranean basin with application in
L. Custódio
:
A. L. Escapa
:
E. Fernandes
:
A. Romano
Institute for Biotechnology and Bioengineering,
Centre of Genomics and Biotechnology (IBB/CGB),
Faculty of Sciences and Technology, University of Algarve,
Ed. 8, Campus of Gambelas,
Faro, Portugal
A. Fajardo
:
R. Aligué
Department of Cell Biology, School of Medicine,
University of Barcelona,
Barcelona, Spain
F. Alberício
Institute for Research in Biomedicine, Barcelona Science Park,
Baldiri Reixac 10,
Barcelona 08028, Spain
F. Alberício
CIBER-BBN, Networking Centre on Bioengineering,
Biomaterials and Nanomedicine, Barcelona Science Park,
Baldiri Reixac 10,
Barcelona 08028, Spain
F. Alberício
Department of Organic Chemistry, University of Barcelona,
Martí i Franqués 1-11,
Barcelona 08028, Spain
N. Neng
:
J. M. F. Nogueira
Department of Chemistry and Biochemistry and Center of
Chemistry and Biochemistry, University of Lisbon,
Faculty of Sciences,
Campo Grande, Ed. C8,
Lisbon 1749-016, Portugal
L. Custódio (*)
CCMAR—Center of Marine Sciences,
Faculty of Sciences and Technology, University of Algarve,
Ed. 7, Campus of Gambelas,
Faro, Portugal
e-mail: lcustodio@ualg.pt
Plant Foods Hum Nutr (2011) 66:78–84
DOI 10.1007/s11130-011-0214-8

the food industry as a source of gum extracted from the
seeds (LBG-E410 ). The germs are used to produce germ
flour with a high content of proteins and highly unsaturated
oil, and employed as a dietetic human food [1, 2]. The
carob bean embryo contains caroubin, a protein with
properties similar to gluten, which confers carob germ flour
the potential as a gluten replacement in cereal-derived foods
for celiac people [3].
Germ flour contains polyphenols, proanthocyanidins,
and ellagi- and gallotannins [4]. These phytoc hemicals
protect the organism from excessive production of free
radicals and reactive oxygen species (ROS), which are
involved in the development of the pathology of numerous
diseases such as Alzheimer and cancer [5]. Since the
prevention of chronic diseases is a more effective strategy
than their treatment, many functional foods are nowadays
aimed at boosting intakes of antioxidants in order to reduce
the risk of diseases linked to oxidative stress. Moreover,
there is increasing evidence that some synthetic antiox-
idants such as butylated hydroxytoluene (BHT) have severe
side effects such as carcinogenicity [6], and thus, studies on
natural antioxidants as nutritional supplements and health
food have gained increasingly greater importance.
To the best of our knowledge, there is no information on
the bioactivity of carob germ flour, and no comparative
studies of the phytochemical contents between genders and/
or cultivars. This study aimed to characterize the phyto-
chemical profile and to determine the antioxidant and the in
vitro cytotoxic properties of germ flour extracts f rom
different female cultivars and hermaphrodite carob trees.
In addition, we assessed the contribution of the major
compound theophylline to the antiproliferative activity of
the crude extract against HeLa cells.
Materials and Methods
Plant Material
Mature fruits were sampled during August and Septem-
ber of 2005 in the Algarve region (Portugal) from six
female Portuguese cultivars of carob tree, namely
Mulata, Galhosa, Aida, Costela/Canela, Gasparinha, and
Preta de Lagos, and from two hermaphrodite trees. Fruits
were manually deseeded, and the seeds were used to
produce germ flour by DANISCO Portugal Industrias de
Alfarroba, LDA.
Preparation of Germ Flour Extracts
The methanol extracts were prepared by Soxhlet extraction
as described previously [7]. For the cell experiments, the
solvent was evaporated and the extracts were re-dissolved
in the appropriate culture medium. All the other assays
were performed with the methanol extracts.
Preparation of Theophylline Solution
The stock solution of t heophylline was prepared on
phosphate-buffered saline (PBS, pH 7.4). Immed iately
before use, working solutions were prepared in culture
medium in concentrations corresponding to the amounts
quantified in the crude extracts.
Phytochemical Evaluation of the Extracts
Total Phenolic Content Total phenolic content was deter-
mined by the Folin–Ciocalteu method [8], and expressed as
gallic acid equivalents in milligrams per gram of extract
(dry weight, DW). Total condensed tannins content was
analyzed by the vanillin assay [9] and expressed as catechin
equivalents in milligrams per gram DW. Total flavonoid
content was estimated by the aluminium chloride method
[10] and expressed as rutin equivalents in milligrams per
gram DW.
High-performance Li quid Chromatography The extracts
(10 mg ml
−1
) were analyzed on a n Agilent 1100 Series
liquid chromatograph with a UV–Vis DAD system
(Agilent Technologies, Germany). Analyses were per-
formed on a Tracer excel 120 ODS-A column (150 mm×
4.0 mm, 5 μm par ticle size , Teknokroma , Spain). The
mobile phase was a mixt ure of 2.5% acetic acid in water
(A) and methanol (B), the applied gradient was 0–50 min:
30–80% B, 50–55 min: 80–30% B, hold for 5 min and the
flow rate was 0.5 ml min
−1
. The anal ys es we re performed
at 25 °C, and the injection volume was 40 μl with a draw
speed of 200 μlmin
−1
. F or the identification of the
phenolic compounds, the retention parameters of each
assay were compared w ith the standard contr ols and the
peak purity with the UV–visible spectral reference data.
The levels of the differ ent compounds were extrapolated
from calibration standa r d curves.
Determination of Antioxidant Activity
The antioxidant activity was evaluated on the extracts at the
concentration of 10 mg ml
−1
. The absorbances were
measured on a Shimadzu UV-160A spectrophotometer.
BHT (E321) was used as the positive control at the con-
centration of 1 mg ml
−1
.
Radical Scavenging Activity Rad ical scavenging activity
(RSA) was evaluated against 1-diphenyl-2-picrylhydrazyl
(DPPH) [11] and 2,2′-azino-bis(3-ethylbenzthiazoline-6-
Plant Foods Hum Nutr (2011) 66:78–84 79

sulphonic acid (ABTS) radicals [12]. Results were expressed
as percent inhibition relative to a blank containing methanol.
Fe
3+
-Reducing Power Assay The Fe
3+
-reducing power was
determined by the method described by Choi et al. [13 ].
The intensity of blue green colour was measured at 700 nm,
with a high absorbance of the reaction mixture indicating an
elevated reducing power.
Evaluation of the in vitro Cytotoxic Activity and Reactive
Oxygen Species Production
Cell Culture and Viability Assays The 4-(3-4-iodophenyl)-
2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate
(WST-1) colorimetric assay was used to assess the effect of
the ext racts on (He La) cells viability [7]. Cells were
cultured in Dulbecco’s modified eagle medium with
1,000 mg ml
−1
of glucose, 10% (v/v) heat-inactivated fetal
bovine serum,
L-glutamine (2 mM), sodium pyruvate
(111 mg l
−1
), penicillin (50 U ml
−1
) and streptomycin
(50 μgl
−1
), and were grown in a incubator at 37 °C, 5.1%
CO
2
in humidified atmosphere. Cells were seeded on 96-
well plates (7×10
3
cell/well), incubated for 24 h and treated
for 6–72 h with the extracts (2.5–40 mg ml
−1
). Then,
20 μl of WST-1 were added to each well, and the
absorbance meas ured 2 h later at 450 nm on a Power
Wave XS spectrophotometer. Results were expressed in
terms of cell viability (%), half maximal inhibitory
concentration (IC
50
,mgml
−1
), and maximal degree of
inhibition Max.,%. To determine the effect of theophylline
on HeLa cells viability, cells were seeded in 96-well plates
and treated with culture medium containing theophylline in
concentrations corresponding to the amounts contained in
the complete extract at the concentration of 10 mg ml
−1
(60–
220 μgml
−1
). After 24–72 h of incubation cell viability was
determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay [14]. Results were ex-
pressed in terms of cell viability (%).
ROS Production The Mulata extract effect on ROS pro-
duction was measured in HeLa cells using 2′,7′-dichlor-
odihydro fluorescein diacetate (DCFH-DA) [15]. Cultivar
Mulata was chosen because it is the most common female
cultivar present in commercial orchards in the southern of
Portugal and the most important i n terms of carob
production. Cells were exposed to the extract (10 mg
ml
−1
, 24 h), trypsinized and washed with PBS. Treated and
control cells were resuspended in PBS containing 10 μM
DCFH-DA at 37 °C for 30 min and then incubated with
4mMH
2
O
2
for 30 min at 37 °C. Relative amount of
intracellular ROS (%) was subjected to evaluation by
fluorescence-activated cell sorting in a flow cytometer
(Coulter XL) and calculated according to the following
equation:
Relative amount of intracellular ROS (%)=(FI
1
/FI
0
)×
100%, where FI
0
was the fluor escence intensity of the
Table 1 Total phenolic content (TPC), total tannin content (TTC), total flavonoid content (TFC), and antioxidant activity measured by the DPPH
and ABTS assays (% inhibition), and Fe
3+
–Fe
2
reducing power assay (absorbance at 700 nm) of germ flour extracts from different origins
Cultivar/tree Yield (%) Phenolics content Antioxidant activity
TPC
a
TTC
b
TFC
c
DPPH ABTS Fe
3+
/Fe
2
Female
Mulata 11.6±1.8 21.9±0.5 cd 4.2±0.4b 11.08±0.0e 57.5±1.2d 78.8±3.0ab 0.9±0.0c
Galhosa 4.3±1.2 32.9±0.9a 6.8±0.4a 22.3±0.1a 89.4±1.1a 85.4±3.6a 1.5±0.1a
Aida 3.2±0.7 19.8±0.6 cd 2.2±0.2c 10.8±0.1f 45.2±1.1e 84.7±3.0a 0.5±0.0d
Gasparinha 5.6±0.7 24.8±0.6bc 1.9±0.2 cd 17.4±0.4c 68.2±0.9b 68.4±6.8b 1.4±0.1ab
Costela/Canela 6.3±1.1 28.1±7.8ab 3.9±0.4b 14.7±0.3d 61.2±0.3c 82.8±3.1a 0.8±0.0c
Mean
d
6.2 25.5 3.8 15.4 64.3 80.0 1.0
Hermaphrodites 7.4±1.8 28.7±0.3ab 4.5±0.2b 18.8±0.2b 86.7±0.8a 65.6±4.1b 1.3±0.0b
BHT
e
–– – – 77.6±1.6 56.2±0.4 3.0±0.0
For each column, statistical analysis was made between cultivars/trees. Values followed by different letters are significantly different at P<0.05
(one-way ANOVA, Duncan’s New Multiple Range Test)
a
mg GAE g
−1
extract (DW; GAE gallic acid equivalents)
b
mg RE g
−1
extract (DW; RE rutin equivalents)
c
mg CE g
−1
extract (DW; CE catechin equivalents)
d
Means of all the values of the cultivars
e
Reference compound, 1 mg ml
−1
80 Plant Foods Hum Nutr (2011) 66:78–84

negative control and FI
1
the fluorescence intensity in the
presence of the extract at an excitation wavelength of
485 nm and an emissi on wavelength of 530 nm.
Statistical Analysis
Values were expressed as mean±standard error of mean
(SEM) for at least three experiments and were subjected to
analysis of variance (ANOVA) to assess treatment differ-
ences using the SPSS statistical package for Windows
(release 16.0, SPSS Inc). Significance between means was
tested by Duncan’s New Multiple Range Test (P=0.05).
Correlations between parameters were investigated using the
correlation and regression program of Microsoft Excel. The
IC
50
values were calculated with the GraphPad Prism 4.
Results and Discussion
Cross-varietal screening tests show that certain genotypes
within a plant species can have widely divergent levels of
antioxidants [16]. Our results support those findings, since
a significant variation in the phenolic profile was observed
between samples from different female cultivars (Table 1).
The extract yield ranged from 3.2% in Aida to 11.6% in
Mulata (Table 1). Hermaphrodites exhibited higher amounts
of phenolic compounds than females (Table 1), except than
Galhosa cultivar. Germ flour extract from Galhosa
exhibited the highest content of the three classes of
phenolic compounds (P<0.001, Table 1).
Gallic acid was present in all samples, while other
compounds were only detected in some cultivars (Table 2).
(+)-Catechin and gentisic acid were only identified in
Galhosa, and chlorogenic, ferulic and vanillin, only in
Mulata (Table 2). These differences can be due to the high
genetic diversity occurring between cultivars of this species
[17]. On the other hand, some compounds could be present
in the extracts, but at concent rations not detectable by the
high-performance liquid chromatography (HPLC) condi-
tions used in this study.
The most ab undant identifie d compound was the
alkaloid theophylline. Theophylline was already detected
in aqueous extracts of carob pods [18] and in this work it
was identified for the first time as the major compound in
Table 2 Phytochemical analysis of germ flour extracts of carob tree from different origins by HPLC-DAD (mg/g extract, DW)
Compound
Cultivar/tree C GEac CLac Ct Fac GA M MG Q R Sac V Total T
Female
Mulata –– 1.5 – 0.2 0.3 –– ––– 0.1 2.1 1.6
Galhosa 3.7 2.0 –––0.6 – 0.3 0.2 –– – 6.8 0.5
Aida –– – –– 0.6 0.5 0.6 0.2 – 0.4 – 2.3 1.0
Gasparinha –– – –– 0.4 1.1 0.5 ––0.4 – 2.4 1.3
Costela/Canela –– – –– 0.4 0.9 0.5 ––0.3 – 2.1 1.3
Mean 3.1 1.1
Hermaphrodites –– – –– 0.5 1.5 0.6 ––0.4 – 3.0 1.4
C (+)-catechin; GEac gentisic acid, CLac chlorogenic acid; Ct catechol; Fac ferulic acid; GA gallic acid; M myricetin; MG methyl gallate; Q
quercetin; R rutin; Sac syringic acid; T theophylline; V vanillin
a
As identified by comparison of the retention parameters with the standard controls and the peak purity with the UV–visible spectral reference data
0 5 10 15 20
Extract concentration (mg ml
-1
)
0 5 10 15 200 5 10 15 20
Cell viability (% of control)
0
20
40
60
80
100
120
'Mulata'
'Galhosa'
'Aida'
'Costela/Canela'
'Gasparinha'
Hermaphrodite
AB C
Fig. 1 Effect of treatment with
extracts from carob tree germ
flour on HeLa cells viability
after 24 h (a), 48 h (b)or72h
(c) of incubation. Values repre-
sent means±SEM (n=3)
Plant Foods Hum Nutr (2011) 66:78–84 81

methanol extracts of carob germ flour. Theophylline is
widely used as a smooth muscle relaxant, and has application
in the treatment of asthma as a bronchodilator. At low dose,
theophylline is an effective add-on therapy to corticosteroids
in controlling asthma, due to its anti-inflammatory action
[19]. Due to the stimulatory influence of theophylline and
other methylxanthines in the central nervous system (CNS)
[20], future work is needed to evaluate the CNS effects of
carob germ flour.
The extracts showed a significant RSA, more pronounced
on the ABTS radical (Table 1). Extracts from Galhosa
exhibited the highest RSA and also the highest reductive
ability (Table 1). Compounds with reducing power indicate
that they are electron donors, and can reduce the oxidized
intermediates of lipid peroxidation processes, therefore they
can act as primary and secondary antioxidants [21]. The
antioxidant activity was correlated with the content of
phenolic compounds, but not with theophylline (data not
shown), and varied according to the extract, probably due to
the different phytochemical profile (Tables 1 and 2). A
positive correla tion between phenolics and antioxi dant
activity is common in natural extracts [22, 23]. Both the
phenolic compounds present in the extract and the synergis-
tic mechanisms between them may be responsible for the
antioxidant activity displayed by carob germ flour.
Extracts from leaves and fruit pulps from carob tree
reduce cell viability of different cancer cell lines [7, 18, 24,
25]. This work provides the first evidence of in vitro
cytotoxic activity of carob germ flour extracts on HeLa
cells. The reduction on cell viability was dose-dependent
and already evident after a 24-h treatment (Fig. 1),
indicating that the phytochemicals in the extracts promptly
triggered a series of cellular events leading to the reduction
of cell viability and/or the induction of cell death. The IC
50
values where generally lower for the 48 and 72 h of
incubation and ranged from 2. 7 mg ml
−1
in Galhosa (72 h)
to 10.3 mg ml
−1
in Gasparinha and Mulata (24 h; Table 3).
It is note worthy that the obtained IC
50
values, while
somewhat high, still indicate some interesting activity.
Thesevaluesarelikelyduetothepresenceoflow
Table 3 Half maximal inhibitory concentration (IC
50
,mgml
−1
) and maximal degree of inhibition (Max., %) of carob germ flour extracts on HeLa
cell line
Cells/incubation Parameter Cultivar
Mulata Galhosa Aida Gasparinha Costela/Canela Hermaphrodites
HeLa
24 IC
50
10.3±0.0b 6.9±0.3b 8.9±0.5a 10.3±0.1b 6.9±1.9b 5.5±0.3b
Max. 52.9±3.9b 85.2 ±1.7b 70.4±0.1b 85.37±4.6a 75.0±1.6b 71.6±5.6b
48 IC
50
5.1±0.9a 4.8±0.1b 7.2±0.0a 7.3±1.1a 5.9±0.3ab 4.5±0.0ab
Max. 71.2±6.1b 95.3±0.3a 88.6±0.5ab 84.7±5.1a 89.8±0.6a 90.5±1.2a
72 IC
50
9.9±0.6b 2.7±0.1a 7.8±0.1a 6.6±0.0a 4.4±0.1a 3.7±0.1a
Max. 91.0±0.0a 94.3±1.4a 95.0±1.2a 93.3±1.6a 95.1±1.0a 94.9±0.0a
For each analysed parameter (IC
50
or Max.), statistical analysis was made between periods of incubation for the same cultivar/tree. For the same
cultivar and parameter, values followed by different letters are significantly different at P<0.05 (one-way ANOVA, Duncan’s New Multiple
Range Test
Relative amount of ROS (%)
0
50
100
150
200
250
Control
H
2
O
2
H
2
O
2
Extract (10 mg ml
-1
)
+
*
***
Fig. 3 Effect of the application of ‘Mulata’ extract on intracellular
ROS production by HeLa cells. Significant differences with control:
*P<0.05, **P<0.01, ***P<0.001
0 50 100 150 200
Cell viability (% of control)
0
20
40
60
80
100
120
24 h
48 h
72 h
Teophylline concentration (µg ml
-1
)
Fig. 2 Effect of treatment with theophylline on HeLa cells viability after
24, 48 and 72 h of incubation. Values represent means±SEM (n=3)
82 Plant Foods Hum Nutr (2011) 66:78–84

concentrations of compounds of interest in the crude
extract, or to compounds with no antioxidant activity. The
enrichment in compounds of interest can be accomplished
by a bioactivity-guided fractionation of the crude extract.
There was a significant gender and cultivar-dependency
on the bioactivity of the extracts. The extracts from Galhosa
and hermaphrodites exhibited a strong cytoto xic activity,
with the lowest IC
50
values (Table 3). The maximal degree
of inhibition for the 24-h period of incubation was highest
for Gasparinha (85.3%), Galhosa (85.2%) and Costela/
Canela (75.0%), followed by the hermaphrodites (71.6%)
and Aida (70.4%), (Table 3).
Numerous studies have show n that antioxidant micro-
nutrients present in food, such as phenolics, can inhibit
carcinogenesis by affecting the molecular events in the
initiation, promotion or progression states [26]. In this
work, positive correlations were found between the total
amount of phenolic compounds identified by HPLC, gallic
acid and the cytotoxic activity on HeLa cells (data not
shown). Moreover, under the experimental conditions and
concentrations tested, theophylline, the mai n compound
detected in the extracts, exerted a cytotoxic activity which
was both time- and concentration-dependent (Fig. 2). When
applied during 24 h, it had no effect on HeLa cells viability
(Fig. 2). However, treatment with concentrations higher
than 100 μgml
−1
for 48 and 72 h resulted in a significant
reduction of cell viability (Fig 2). These results suggest the
contribution of the phenolic compounds and theophylline to
the cytotoxic activity of the samples towards HeLa cells.
In this work, it was evaluated the Mulata extract effect
on ROS production after challenging HeLa cells with H
2
O
2
to produce the oxidative stress. As shown in Fig 3, the
intracellular ROS accumulation resulting from H
2
O
2
expo-
sure was significantly increased in the presence of the
extract (207.5±1.6%, P<0.05), compared with cells in the
presence of H
2
O
2
without extract (110.7±9.8%, P<0.05;
Fig. 3). This may be related with the induction of early
apoptosis. ROS can act as anticancer, namely by the
promotion of cell-cycle stasis, senescence, apoptosis and
necrosis, or as pro-cancer, stimulating cell proliferation,
causing DNA mutations, and promoting genetic instability
[27]. Additional studies are needed to clarify the mecha-
nism of actio n of methanol extracts from carob germ flour,
namely through the assessment of its effect on cell-cycle
progression, its ability to activate caspases, or mitochon -
drial and DNA damages.
Conclusions
This work shows that carob germ flour is a source of
compounds with antioxidant and in vitro cytotoxic activi-
ties, and that those activities are strongly dependent on the
cultivar. The female cultivar Galhosa revealed to be the best
source of bioactive compounds. Additional experiments are
needed in order to identify and characterize the bioactive
compounds present in the extracts, namely through the bio-
guided fractionation and isolation of pure compounds.
Acknowledgements This work was partially supported by CICYT
(CTQ2006-03794/BQU), Instituto de Salud Carlos III
(CB06_01_0074 and PI060624), the Generalitat de Catalu nya
(2005SGR 00662), the Institute for Research in Biomedicine, and
the Barcelona Science Park. L. Custódio thanks to the Portuguese
Foundation for Science and Technology (FCT) for a post-doctoral
grant (grant SFRH/BPD/20736/2004). The authors thank DANISCO
Portugal Industrias de Alfarroba, LDA for providing the germ flour
samples.
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