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Fucoxanthin, the constituent of Laminaria japonica, triggers AMPK-mediated cytoprotection and autophagy in hepatocytes under oxidative stress

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Background: Laminaria japonica has frequently been used as a food supplement and drug in traditional oriental medicine. Among the major active constituents responsible for the bioactivities of L. japonica, fucoxanthin (FX) has been considered as a potential antioxidant. This study was conducted to examine the effects of L. japonica extract (LJE) or FX against oxidative stress on hepatocytes and to elucidate the overall their cellular mechanisms of the effects. Methods: We constructed an in vitro model with the treatment of arachidonic acid (AA) + iron in HepG2 cells to stimulate the oxidative damage. The cells were pre-treated with LJE or FX for 1 h, and incubated with AA + iron. The effect on oxidative damage and cellular mechanisms of LJE or FX were assessed by cytological examination and several biochemical assays under conditions with or without kinase inhibitiors. Results: LJE or FX pretreatment effectively blocked the pathological changes caused by AA + iron treatment, such as cell death, altered expression of apoptosis-related proteins such as procaspase-3 and poly (ADP-ribose) polymerase, and mitochondria dysfunction. Moreover, FX induced AMPK activation and AMPK inhibitor, compound C, partially reduced the protective effect of FX on mitochondria dysfunction. Consistent with AMPK activation, FX increased the protein levels of autophagic markers (LC3II and beclin-1) and the number of acridine orange stained cells, and decreased the phosphorylation of mTOR and simultaneously increased the phosphorylation of ULK1. And the inhibition of autophagy by 3-methylanine or bafilomycin A1 partially inhibited the protective effect of FX on mitochondria dysfunction. Conclusion: These findings suggest that FX have the function of being a hepatic protectant against oxidative damages through the AMPK pathway for the control of autophagy.
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R E S E A R C H A R T I C L E Open Access
Fucoxanthin, the constituent of Laminaria
japonica, triggers AMPK-mediated
cytoprotection and autophagy in
hepatocytes under oxidative stress
Eun Jeong Jang
1
, Sang Chan Kim
1
, Ju-Hee Lee
1,2
, Jong Rok Lee
1
, Il Kon Kim
3
, Su Youn Baek
1*
and Young Woo Kim
1,3*
Abstract
Background: Laminaria japonica has frequently been used as a food supplement and drug in traditional oriental
medicine. Among the major active constituents responsible for the bioactivities of L. japonica, fucoxanthin (FX) has
been considered as a potential antioxidant. This study was conducted to examine the effects of L. japonica extract (LJE)
or FX against oxidative stress on hepatocytes and to elucidate the overall their cellular mechanisms of the effects.
Methods: We constructed an in vitro model with the treatment of arachidonic acid (AA) + iron in HepG2 cells to
stimulate the oxidative damage. The cells were pre-treated with LJE or FX for 1 h, and incubated with AA + iron. The
effect on oxidative damage and cellular mechanisms of LJE or FX were assessed by cytological examination and several
biochemical assays under conditions with or without kinase inhibitiors.
Results: LJE or FX pretreatment effectively blocked the pathological changes caused by AA + iron treatment, such as cell
death, altered expression of apoptosis-related proteins such as procaspase-3 and poly (ADP-ribose) polymerase, and
mitochondria dysfunction. Moreover, FX induced AMPK activation and AMPK inhibitor, compound C, partially reduced the
protective effect of FX on mitochondria dysfunction. Consistent with AMPK activation, FX increased the protein levels of
autophagic markers (LC3II and beclin-1) and the number of acridine orange stained cells, and decreased the
phosphorylation of mTOR and simultaneously increased the phosphorylation of ULK1. And the inhibition of autophagy by
3-methylanine or bafilomycin A1 partially inhibited the protective effect of FX on mitochondria dysfunction.
Conclusion: These findings suggest that FX have the function of being a hepatic protectant against oxidative damages
through the AMPK pathway for the control of autophagy.
Keywords: Fucoxanthin, Oxidative stress, AMPK, Autophagy, AMPK/mTOR/ULK-1 pathway
Background
Laminaria japonica, one of the most well known brown
seaweeds, is referred to as Dashimain Korean,
Kombuin Japanese, and Haidaiin Chinese. L. japonica
is widely used as a food supplement, as well as a drug for
treatment of various diseases [1]. L. japonica has abundant
bioactive components, including polyphenols, pigments,
polysaccharides, minerals and amino acids [1]. Among
bioactive compounds in L. japonica, fucoxanthin (FX), a
marine carotenoid, has remarkable biological properties,
including anti-cancer, obesity and inflammation [14]. FX
has attracted much attention as a potential antioxidant
given its unique chemical structure (Fig. 2a), which in-
cludes an allenic bond, epoxide group, and hydroxyl group
[5]. Liu et al. (2011) reported that FX significantly recov-
ered cell proliferation and increased the levels of glutathi-
one and decreased intracellular reactive oxygen species
(ROS) induced by ferric nitrilotriacetate [6]. In recently,
Seo et al. (2016) showed that FX inhibited lipid
* Correspondence: rhodeus@dhu.ac.kr;ywkim@dhu.ac.kr
Equal contributors
1
College of Oriental Medicine, Daegu Haany University, Gyeongsan,
Gyeongsangbuk-do 38610, South Korea
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97
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accumulation and ROS formation by controlling adipo-
genic and lipogenic factors and ROS-regulating enzymes
during differentiation in 3 T3-L1 adipocytes [7]. It is indi-
cated that FX can effectively protect against hepatotoxicity
by reducing intracellular ROS, associated with the antioxi-
dant effects of FX.
ROS is a group of molecules including superoxide
anion, hydroxyl radical and hydrogen peroxide, mainly
produced in the mitochondria [8]. However, excess ROS
can be involved in oxidative stress that destroys the
structure of vital biomolecules, potentially leading to
cellular dysfunction and remodeling [9]. Oxidative stress
is known to activate the AMP-activated protein kinase
(AMPK) signaling system in neuronal, heart, muscle,
pancreatic and liver cells [10,11]. Interestingly, AMPK
is known to be involved in ROS-induced autophagy that
promotes cell survival in response to cellular stress such
as malnutrition, hypoxia or ischemia [12]. Indeed, oxy-
gen and nutrient deprivation induce the activation of
AMPK leading to autophagy by inhibition of mTORC1
and phosphorylation of ULK1 [13,14].
Autophagy is an important cell pathway for cell
homeostasis and survival by removing damaged organ-
elles and intracellular microbial pathogens [15]. Hepato-
cytes may be particularly dependent on the underlying
autophagy for normal physiological function due to their
high biosynthetic activity. In addition, autophagy plays a
crucial role in the non-alcoholic and alcoholic liver dis-
eases, drug-induced hepatic damage, viral hepatitis, fi-
brosis, liver cancer and hepatic ischemia reperfusion
injury [1517]. In liver ischemia reperfusion injuries, au-
tophagy provides a prosurvival activity allowing the cell
to cope with nutrient starvation and anoxia [16]. During
hepatitis B or C infection, the level of autophagy is typic-
ally increased to promote viral growth [17]. In hepato-
cellular carcinoma, the level of autophagy is thought to
be involved in both tumorigenesis and tumor suppres-
sion [18,19].
In this regard, we tested whether L. japonica and FX
alleviated hepatic oxidative stress in an in vitro model,
HepG2 cells established by arachidonic acid (AA) + iron.
Specifically, we explored the abilities of FX in regulation
of autophagy and the underlying molecular mechanisms
of their effects.
Methods
Reagents
AA and Compound C (C.C) were purchased from
Calbiochem (San Diego, CA, USA). Anti-phospho-ACC,
phospho-LKB1, procaspase-3, PARP, Bcl
XL,
LC3 I/II,
beclin-1, AMPK, and phospho-AMPK antibodies were
obtained from Cell Signaling Technology (Beverly, MA,
USA). Bal-A1 was purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). Horseradish peroxidase-
conjugated goat anti-rabbit, rabbit anti-goat, and goat
anti-mouse IgGs were obtained from Zymed Laboratories
(San Francisco, CA, USA). FX, acrydine orange hemi zinc
chloride salt, 3-methyladenine (3-MA), anti-ß-actin anti-
body and other reagents were purchased from Sigma-
Aldrich (St. Louis, MO, USA).
Preparation of the L. japonica extract (LJE)
L. japonica was purchased from Daewon pharmacy
(Daegu, Korea), which is standardized with a standard herb
of L. japonica in Korea Food and Drug Administrations.
The L. japonica (100 g) were extracted as previously
described [20,21]. The yield of lyophilized LJE was esti-
matedtobe1.19%basedonthedriedweight.
Cell culture
HepG2 cells, a human hepatocyte-derived cell line, were
provided by American Type Culture Collection (Rockville,
MD, USA), and cultured as previously described [20]. To
simulate oxidative stress, cells were incubated with 10 μM
AA for 12 h, followed by exposure to 5 μM iron for 1 h.
The cells were treated with FX or LJE for 1 h prior to the
incubation with AA at the indicated doses.
Cell viability assay
The cells were plated at a density of 1 × 10
5
cells per well
in a 48-well for 24 h as previously described [20]. The
media was incubated with 0.25 mg/ml MTT for 2 h, and
formazan crystals were dissolved with the addition of
200 μl DMSO.
Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assay
The TUNEL assay was performed using the DeadEnd
Colorimetric TUNEL System, according to the manufac-
turers instruction. The samples were washed and exam-
ined under light microscope.
Western blot analysis
The cells were plated at a density of 5 × 10
5
cells per well in
a 6-well plate for 24 h. After the treatment designated, cells
were lysed in RIPA buffer (Thermo Scientific, Rockford, IL,
USA) as previously described [20,21]. The protein bands
were detected using Fusion Solo scanning system (Vilber
Lourmat, Paris, France), and quantified using Image J ver
1.42 software (NIH, Bethesda, USA).
Measurement of ROS production
DCFH-DA, a cell-permeable non-fluorescent probe, has
been used as a substrate for quantitation of intracellular
oxidant production in HepG2 cells [21]. After treatment
of reagents, cells were stained with 10 μM DCFH-DA
for 30 min at 37 °C. The fluorescence intensity in the
cells was measured at an excitation/emission wavelength
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 2 of 11
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of 485/535 nm, using in the cells measured in a micro-
plate reader.
Determinant of glutathione (GSH) content
Intracellular GSH content was quantified using a commer-
cial GSH BIOXYTECH GSH-400 kit (Oxis International,
Portland, OR) according to the manufacturersprotocol,
and the absorbance level was measured at 405 nm.
Flow cytometric analysis of ΔΨm
ΔΨm was measured using rhodamine123 (Rh123).
Following treatment, cells were stained with 0.05 μg/ml
of Rh123 for 1 h and then harvested by trypsinization.
The change in ΔΨm was monitored using a FACS flow
cytometer (Partec, Münster, Germany). In each analysis,
a total of 10,000 events were recorded as previous
described [20].
Acridine orange (AO) staining
HepG2 cells were plated on 18-mm cover glasses and in-
cubated for 24 h to reach at approximately 70% conflu-
ence. They were then incubated either in the presence
or absence of 30 μM FX, washed twice with PBS and
fixed with ice-cold 4% paraformaldehyde for 10 min at
room temperature. Subsequently, the cells were stained
with AO (1 μg/ml) for 15 min at room temperature,
washed, and examined under a fluorescence microscope
(Nikon, Japan).
Profiling the content of fucoxanthin by ultra performance
liquid chromatography (UPLC)
Water ACQUITYTM ultraperformance LC system
(USA) was used to assess UPLC analysis as previously
described [20]. Waters ACQUITYTM BEH C18 column
(1.7 μm, 2.1 mm × 100 mm) was used as Waters
ACQUITYTM PDA and HPLC Column, and the fuco-
xanthin was analyzed at 330 nm. The standard fucoxan-
thin was melted by methanol and diluted to make
solution containing 1 μg/ml. L. japonica 1 g was also
added with methanol 10 ml, and then sonication was
perfomed for 3 h. After then, it was filtered through a
0.2 μm filter (Nalgene, NY, USA). A mobile phase was a
mixed liquid of the acetonitrile and water, and the ana-
lysis condition was as in Table 1. The sample was
injected with 2 μl, and a flow rate was 0.4 ml/min.
Statistical analysis for study
GraphPad Prism software version 5.01 (Graph Pad Software,
La Jolla, CA) was used for all statistical analyses as previ-
ously described [20,21]. Significance levels were calculated
by repeated measures of ANOVA with the Dunnett post
hoc test under 95% confidence interval. Data were presented
as the mean with standard deviation (mean ± S.D.). Within
figures, the Pvalues were displayed with asterisks
(***P < 0.001, **P < 0.01, *P < 0.05).
Results
L. japonica Extract (LJE) decreases AA + iron induced
cytotoxicity in HepG2 cells
An MTT assay for cell viability indicated that LJE pre-
treatment (3, 10, 30, and 50 μg/ml) significantly pro-
tected cells from the potential injury induced by AA +
iron. Since the maximum cell viability was achieved at
30 μg/ml of LJE, the same concentration was applied in
subsequent experiments (Fig. 1a). In western blot ana-
lysis, treatment of AA + iron markedly induced
decreases in the protein levels of procaspase-3 and
Bcl
XL
, verifying AA + iron induction of apoptosis, which
was completely blocked by LJE pretreatment (Fig. 1b).
Morphological examination by light microscopy and
TUNEL assay (Fig. 1c) confirmed the cytoprotective
effect of LJE against the synergized toxicity of AA + iron.
After treatment with LJE, positive staining located in the
nucleus by AA + iron were apparently decreased
(Fig. 1c). To further examine the antioxidative effects of
LJE, we measured the contents of GSH and ROS. The
intracellular concentration of GSH was substantially
decreased by AA + iron, but was recovered by LJE treat-
ment. In contrast, treatment with LJE alone had no ef-
fects on cellular GSH levels (Fig. 1d). The ROS
generation assay using DCFH-DA indicated that LJE
treatment effectively abrogated increases in ROS produc-
tion caused by AA + iron (Fig. 1e). The effect of LJE on
AA + iron-induced loss of mitochondrial membrane
potential (ΔΨm) monitored by FACS analysis of Rho123
staining (Fig. 1f ). Rho123 fluorescence intensity was not
significantly altered in LJE-treated cells compared to
untreated controls, though AA + iron markedly reduced
rhodamine fluorescence, indicating the loss of ΔΨm
(Fig. 1f). LJE treatment significantly restrored the loss of
ΔΨm caused by AA + iron (Fig. 1f ).
Table 1 Solvent gradient for the analysis fucoxanthin in L. japonica
Time (min) 0.1% FA/
Water (%)
0.1% FA/
Acetonitrile (%)
Flow rate
(ml/min)
0 98 2 0.40
1 98 2 0.40
2 90 10 0.40
4 70 30 0.40
7 50 50 0.40
9 30 70 0.40
10 10 90 0.40
12 0 100 0.40
14 98 2 0.40
16 98 2 0.40
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a
c
d
f
e
b
0
20
40
60
80
100
120
Vehicle - 3 10 30 50
Cell viability (%)
-actin
Procaspase-3
BclXL
Vehicle _ LJE LJE (30µg/ml)
AA+iron
(LJE, µg/ml)
0
20
40
60
80
Vehicle - LJE LJE
TUNEL-positive cells (%)
AA+iron
**
Vehicle
LJE
AA+iron
-
**
** **
NS NS
**
AA+iron
0
10000
20000
30000
40000
Vehicle - LJE LJE
ROS product
AA+iron
0
30
60
90
120
150
Vehicle - LJE LJE
GSH (nmol/mg protein)
AA+iron
** **
** **
(30µg/ml)
(30µg/ml) (30µg/ml)
Counts
Rhodamine 123
Vehicle AA+iron AA+iron+LJE(30µg/ml) LJE (30µg/ml)
0
30
60
90
120
Vehicle - LJE LJE
Loss of m (%)
AA+iron
** **
(30µg/ml)
Fig. 1 L. japonica extract (LJE) decreases AA + iron induced cytotoxicity in HepG2 cells. HepG2 cells were incubated with indicated dose of LJE
for 1 h and later treated with 10 μM AA for 12 h, being followed by exposure to 5 μM iron for 3 h. (a) Cell viability was assessed by the MTT
assay. (b) Expression of proteins associated with apoptosis was determined by western blot analysis. Equal protein loading was verified by β-actin.
(c) The levels of apoptosis in each groups examined by TUNEL assay. Representative images show apoptosis of HepG2 cells in vehicle control, LJE
treated, AA + iron treated, and AA + iron treated with LJE groups (left). Percentage of TUNEL+ cell nuclei calculated relative to total number of
cell nuclei (right). (d) Cellular GSH content was assessed in cells by using GSH assay kit. (e) Cellular reactive oxygen species production was moni-
tored by measuring intensity of dichloro fluoresce in fluorescence. (f)ΔΨm depolarization monitored by FACS analysis of Rh123 staining. Relative
proportions of low Rh-123 intensity (RN1 fractions) are expressed as the mean ±S.D. of three separated experiments. For panel from A to E, data
represent the mean ± S.D. for the four replicates. ** p< 0.01
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Fucoxanthin (FX) ameliorates AA + iron-induced
cytotoxicity
Next, we determined the effects of FX against oxidative
stress induced by AA + iron (Fig. 2a). FX treatment inhib-
ited death of cell induced by AA + iron, and this decrease
in cell viability was recovered by pre-treatment with 30 μM
of FX (Fig. 2b). In western blot analysis, cleavage of PARP
and caspase-3 were strongly observed in AA + iron-treated
cells, which were blocked by FX pretreatment (Fig. 2c). FX
treatment significantly inhibited the change in ΔΨmcaused
by AA + iron (Fig. 2d). These results indicate that FX
remarkably suppressed AA + iron-induced collapse of
ΔΨm, consequently protecting liver cells.
FX-induced the activation of AMPK alleviates cell damage
by oxidative stress
Stimulation of FX (30 μM) markedly induced the phos-
phorylation of AMPK (Fig. 3a). This compound also in-
duced the phosphorylation of LKB1, an upstream kinase
of AMPK, and ACC, a primary downstream target of
AMPK (Fig. 3a). To determine the role of AMPK in pro-
tection of HepG2 cells by FX, we measured ΔΨm levels
after treating with a chemical inhibitor of AMPK, C.C.
C.C inhibited the protective effect of FX on AA + iron-
induced the loss of ΔΨm in HepG2 cells (Fig. 3b). Col-
lectively, these results suggest that FX activates the
LKB1-AMPK signaling pathway, and that this activation
0
20
40
60
80
100
120
Vehicle - 3 10 30 60
Cell viability (%)
AA+iron
**
**
FX (µM)
Fucoxanthin
pro-PARP
cleaved form
cleaved form
pro-caspase 3
-act in
Vehicle - FX FX
AA+iron
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
3
3.5
4
Vehicle FX FX
AA+iron
Relative protein level
(fold of vehicle)
Cleaved
PA R P
Cleaved
caspase 3
**
**
Relative protein level
(fold of vehicle)
0
20
40
60
80
Vehicle - FX FX
AA+iron
** **
Loss of m (%)
** **
NS
(30µM)
(30µM)
(30µM)
a
c
d
b
Fig. 2 Fucoxanthin (FX) ameliorates AA + iron-induced cytotoxicity. HepG2 cells were incubated with indicated dose of FX for 1 h and later treated with
10 μM AA for 12 h, being followed by exposure to 5 μMironfor3h.(a) The chemical structure of FX. (b) The effects of FX on cell viability was assessed by
the MTT assay. (c) Expression of proteins associated with apoptosis was determined by western blot analysis. Equal protein loading was verified by β-actin.
(d)ΔΨm depolarization monitored by FACS analysis of Rh123 staining. Relative proportions of low Rh-123 intensity (RN1 fractions) are expressed as the
mean ± S.D. of three separated experiments. For panel b,cand d, data represent the mean ± S.D. for the four replicates. ** p<0.01
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 5 of 11
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of beneficial molecules is responsible for FXs inhibition
of mitochondria damage induced by oxidative stress.
FX triggers AMPK-dependent cytoprotective autophagy
Treatment with 30 μM FX upregulated beclin-1 and
promoted the conversion of LC3 I to LC3 II as com-
pared to the control (Fig. 4a). The AVOs were clearly
observed in the HepG2 cells (red fluorescence) following
treatment with FX (Fig. 4b). As shown in Fig. 4c, inhib-
ition of AMPK activity by C.C markedly attenuated FX-
induced accumulation of beclin-1, suggesting that
AMPK is critical in the regulation of FX-induced au-
tophagy. In addition, western blotting revealed that FX
rapidly downregulated the phosphorylation of mTORC1
(Ser2448), which is known to negatively modulate au-
tophagy and be inhibited by AMPK (Fig. 5a). In contrast,
the phosphorylation levels of ULK1 (Ser555) was in-
creased during FX treatment in time-dependent manner
(Fig. 5a). Furthermore, we manipulated autophagy activ-
ity using 3-MA and Baf-A1 to suppress autophagy.
FACS analysis of ΔΨm showed that 3-MA and Baf-A1
partially blocked the effect of FX on mitochondrial pro-
tection (Fig. 5b).
Discussion
In our results, LJE or FX ameliorated oxidative damage
induced by AA + iron on hepatocytes, as confirmed by
the inhibition of cell death and the restorating the loss
of ΔΨm. We also demonstrated that these hepatoprotec-
tive effects of FX can be attributed to the function of au-
tophagy via the AMPK/mTORC1/ULK-1 axis.
To determine the capacity of LJE or FX to reduce oxi-
dative damage, we employed an in vitro model, HepG2
cells treated with AA + iron. In liver, the level of iron is
tightly regulated by the control of absorption, storage
and recycling, which is critical for the protection of liver
tissues as well as other organ tissues from iron-induced
cellular damages [21]. However, a chronic increase of
iron level in liver can result in excess ROS production
and liver injury, such as steatohepatitis, fibrosis,
a
b
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
p-ACC
p-LKB1
p-AMPK
-act in
LKB1
0
0.5
1
1.5
2
2.5
3
Relative level of p-LKB1
(fold ofvehicle)
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
0
0.4
0.8
1.2
1.6
Relative level of p-AMPK
(foldof vehicle)
FX
Vehicle 10’ 30’ 1 3 6 (H)
*
0
20
40
60
80
100
C. C
**
*
Loss of m (%)
- + - + - +
AA+iron - - + + + +
FX (30µM) - - - - + +
Fig. 3 FX-induced the activation of AMPK alleviates cell damage by oxidative stress. (a) FX induces phosphorylation of the proteins associated with
AMPK pathway, ACC, LKB1 and AMPK. Western blot analyses were performed with the lysates of cells that had been treated with 30 μM FX for the
indicated time period. β-actin served as a loading control. Protein levels were presented as relative band intensities to control (vehicle treated) group.
Results represent the mean ±S.D. for three separate experiments. * p<0.05; **p< 0.01; *** p<0.001.(b) The effect of FX to restore ΔΨmwasrevered
by C.C. Following treatment with 10 μM C.C for 1 h, cells were incubated with FX and/or AA + iron, and ΔΨm was evaluated with Rh123 stain by FACS.
Data represent the mean± S.D. for four replicates. * p<0.05; ** p<0.01
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cirrhosis, and hepatocellular carcinoma [22]. The release
of AA can also be induced by an increase in oxidative
stress that originates from excessive levels of iron [23].
Although prostaglandins, essential components in cellu-
lar protection, are produced from AA, excessive AA can
induce extremely high levels of cellular and mitochon-
drial ROS, negatively influencing the functions of several
processes related to mitochondrial respiration [23]. The
combinatorial treatment of AA and iron can thus reduce
cell viability, and this treatment may be used to test the
potential of cytoprotective agents targeting mitochondria
against severe oxidative stress. In this study, AA + iron
successfully induced cell death, production of ROS and
damage of mitochondria in HepG2 cells. However,
pretreatment of LJE or FX significantly blocked the abil-
ity of AA + iron to induce similar detrimental effects in
HepG2 cells.
Recent studies have shown that AMPK serves as a key
regulator of hepatocytes viability under oxidative stress
[10,11,23]. Indeed, many natural compounds, such as
resveratrol, sauchinone, and isoliquiritigenin, have been
reported to protect hepatocytes by inhibiting production
of ROS and mitochondrial dysfunction through activa-
tion of AMPK [2426]. In our study, FX upregulated the
phosphorylation of AMPK, ACC (the downstream
target) and LKB1 (the essential upstream kinase) in he-
patocytes. Furthermore, the inhibition of AMPK using
compound C reduced the beneficial effect of FX on
LC3 I
LC3 II
Beclin-1
-actin
F X (30µM) Control
Relative level of LC3 II
(fold of v e h ic le)
Re la ti ve lev e l of beclin-1
(fold of vehicle)
0
0.4
0.8
1.2
1.6
2
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
0
0.4
0.8
1.2
1.6
Vehicle 10’ 30’ 1 3 6 (H)
FX
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
***
C.C (µM) - 10 - 10
FX (µM) - - 30 30
Beclin-1
-act in
*** ***
a
b
c
Fig. 4 The effect of FX on autophagy induction in HepG2 cells. (a) FX induces time-dependent activation of the autophagy related proteins, LC3II and
Becline-1. Western blot analyses were performed on the lysates of cells that had been treated with 30 μM FX for the indicated time period. β-actin served
as a loading control. Protein levels were presented as relative band intensities to control (vehicle treated) group. Results represent the mean ± S.D.forfour
separate experiments. ** p< 0.01; *** p< 0.001. (b) The pictures of the fluorescence micrographs show the formation of AVOs resulting from the treatment
of FX in HepG2 cells. Cells were incubated either in the presence or absence of 30 μM FX for 6 h and were stained with AO. The presence of AVOs was
indicated by the red fluorescence. (c) Inhibition of FX-induced autophagy by C.C. Western blot analysis of Beclin-1 were performed with lysates of HepG2
cells that had been pretreated with 10 μM C.C for 1 h being followed by exposure to 30 μMFXfor1h
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 7 of 11
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mitochondria. Those results partially suggest that FX
protects cells through activation of AMPK.
It is now widely accepted that AMPK induce autophagy,
in turn, serving to reduce oxidative damage [1214]. Au-
tophagy can suppress cell death by eliminating damaged
organelles or unnecessary cellular components formed
from a variety of stresses, thereby playing adaptive roles to
protect organisms against infections, cancer, neurodegen-
eration, aging, and heart diseases [1518,27]. The process
of autophagy involves formation of double membrane
vesicles (autophagosome) that enwrap portions of the
cytoplasm [24]. To detect the development of AVOs,
HepG2 cells treated with FX were stained with acridine
orange. Our results demonstrated that the bright red
fluorescence significantly increased after FX treatment,
indicating the development of AVOs. Western blotting
studies also demonstrated that FX induced HepG2 cell au-
tophagy, as shown by the conversion of LC3B-I in LC3B-
II and the expression of Beclin-1, indicators of autophagy
[24,28]. Furthermore, AMPK inhibition by a chemical in-
hibitor of AMPKα, C.C abolished the increased protein
level of beclin-1 by FX. Consequently, our current findings
suggest that the activation of AMPK by FX be involved in
induction of autophagy in HepG2 cells.
AMPK activation promoted autophagy through direct
activation of ULK1 and inhibition of mTORC1, a nega-
tive regulator of autophagy [14]. mTORC1 is important
in the autophagy and its activity inhibits autophagy by
ULK1 phosphorylation, which induce disassociation be-
tween ULK1 and AMPK [14]. In this study, we observed
a significant increase in the levels of ULK1 and AMPK
phosphorylation in response to FX. Thus, our findings
indicate a model in which FX induces autophagy by acti-
vating the AMPKULK1mTORC1 axis. Finally, present
study showed that 3-MA and Baf-A1, the autophagy in-
hibitors, partially reversed the inhibitory effect of FX on
AA + iron-induced mitochondrial membrane potential
depolarization in HepG2 cells, suggesting the involve-
ment of AMPK-induced autophagy as the hepatoprotec-
tive activity of FX in HepG2 cells.
Although there are various component in L. japonica,
in this study, we confirmed the content of FX (mass
p-mTOR
(Ser2448)
ULK1
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
-actin
mTOR
p-ULK1
(Ser 555)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Relative levelof p-mTOR
(fo ld o fvehi cle )
*
***
FX
Vehicle 10’ 30’ 1 3 6 (H)
0
0.5
1
1.5
2
2.5
3
Relative leve l of p-ULK1
(foldofvehicle)
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
0
20
40
60
80
100
-+-+-+
++ ++ ++
FX
(30µM)
AA+iron
Veh i c l e 3MA Baf-A1
*** *** **
*** ***
Loss of m (%)
a
b
Fig. 5 The role of AMPK activation by FX in autophagy induction. (a) Western blot analysis for the phosphorylated level of mTOR (Ser2448) and ULK1
(Ser555) in HepG2 cells treated with 30 μM FX for indicated time period. Results represent the mean ± S.D. for four separate experiments.
*p< 0.05; ** p< 0.01; *** p<0.001.(b) The effect of FX to restore ΔΨm was revered by 3-MA and Baf-A1. After 3-MA and Baf-A1 treatment (5 μMfor
1 h, respectively), cells were incubated with FX for 1 h, being followed by the addition of AA (for 12 h) + iron (1 h). Data represent the mean ± S.D. for
three replicates. ** p< 0.01; *** p<0.001
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 8 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 6 UPLC chromatogram of fucoxanthin standard (a) and fucoxanthin in L. japonica (b). The peak represents fucoxanthin (330 nm)
Arachidonic acid
+ Iron
Oxidative
stress
Intracellular ROS
Mitochondrial
dysfunction
Fucoxanthin
LKB1
P
AMPK
P
mTOR
ULK1
AutophagyApotopsis
Laminaria japonica
Fig. 7 Schematic diagram showed that FX induces AMPK-mediated autophagy contributing to ameliorates oxidative stress in HepG2 cells
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 9 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
accuracy; 28.197 ppm) in L. japonica (Fig. 6). Recently,
some report also showed that FX is known to significantly
inhibit on the proliferation of HepG2 cells [29]. The result
showed that both the protein degradation and transcrip-
tional repression was responsible for cyclin D suppression
by FX in HepG2 cells by inducing G1 arrest as mediated
with GADD45A and MAPK pathway [29]. But, in the
present study, FX showed a significantly protective effect
on HepG2 cells against AA + iron-induced oxidative in-
jury. Therefore, the active compound in the L. japonica
and FX in the aspect of cell protection as well as their
machanism remains to be further established.
Conclusion
Our results suggested that FX could protect hepatocytes
against AA + iron-induced oxidative stress and trigger
autophagy, which is likely associated with the LKB1-
AMPKαsignaling pathway (Fig. 7). The current study
also showed that FX or Laminaria japonica likely con-
tributed to further understanding of its potential use as
a hepatic protectant and nutraceutical.
Abbreviations
3-MA: 3-methyladenine; AA: Arachidonic acid; ACC: Acetyl-CoA carboxylase;
AMPK: AMP-activated protein kinase; ATG: Autophagy-related proteins; AVO: Acidic
vesicular organelle; Bal-A1: Bafilomycin A1; Bcl-xL: B-cell lymphoma-extra large;
C.C: Compound C; DCFH-DA: 2,7-Dichlorofluorescein diacetate; DMSO: Dimethyl
sulphoxide; FBS: Fetal bovine serum; FX: Fucoxanthin; GSH: Glutathione;
LC3: Microtubule-associated protein 1 light chain 3; LJE: L. japonica extract;
LKB1: Liver kinase B1; mTOR: mammalian target of rapamycin; MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PARP: Poly (ADP-ribose)
polymerase; PBS: Phosphate-buffered saline; Rh123: Rhodamine 123; ROS: Reactive
oxygen species; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end
labeling; ΔΨm: mitochondrial membrane potential
Funding
This work was supported by the National Research Foundation of Korea (NRF)
grant funded by the Korea Government [MSIP] (No.2015K1A3A1A59069800)
and (No. 2017R1D1A3B03027847), and the NRF grant funded by the Korea
government [MSIP] (No. 2012R1A5A2A42671316), and and also by the Grant
K18102 awarded to Korea Institute of Oriental Medicine (KIOM) from Korea
Ministry of Education, Science and Technology (MEST).
Availability of data and materials
The datasets generated and/or analyzed during this study are available from
the corresponding author on reasonable request.
Authorscontributions
E.J.J., S.C.K., J.H.L. and S.Y.B. conducted research and cell experiments of
fucoxanthin and L. japonica. J.R.L. analyzed the contents of fucoxanthin in L.
japonica. S.C.K. and S.Y.B. helped the writing the paper and the analysis of
data. E.J.J., J.H.L., S.Y.B., I.K.K. and Y.W.K designed research and wrote the
paper. S.C.K., S.Y.B. and Y.W.K. supported financial funding. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
PublishersNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
College of Oriental Medicine, Daegu Haany University, Gyeongsan,
Gyeongsangbuk-do 38610, South Korea.
2
College of Korean Medicine,
Dongguk University, Gyungju, Gyeongbuk 38066, South Korea.
3
Kyungpook
National University, Daegu 41566, South Korea.
Received: 31 August 2017 Accepted: 8 March 2018
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... Neste ensaio, a fucoxantina reduziu a produção de ROS intracelulares, os danos no DNA e aumentou os níveis de glutationa, desempenhando um importante papel na defesa celular contra oxidantes e na manutenção da homeostase redox celular altamente relacionada com a prevenção de processos apoptóticos [180]. Seguindo essa tendência, a fucoxantina demonstrou inibir a acumulação de lípidos e a formação de ROS em adipócitos de ratos 3T3-L1 [156]. A fucoxantina também exibiu efeitos antioxidantes em linhas celulares humanas com resultados semelhantes aos observados em modelos animais. ...
... Ácido araquidónico e ferro foram usados para estimular danos oxidativos e apoptose em células hepáticas (Hep G2) pré-tratadas com fucoxantina extraída de L. japonica. Os resultados demonstraram que o pré-tratamento com fucoxantina (30 µM/mL) permitiu reduzir a morte celular, disfunção mitocondrial e danos oxidativos e desencadeou a autofagia através da activação da proteína quinase AMP-activada (AMPK) que promove a remoção de componentes celulares danificados representando uma via fundamental para a homeostase celular [156]. Efeitos concordantes com os descritos acima foram observados em células endoteliais da veia umbilical humana expostas a lesões em lipoproteínas de baixa densidade oxidadas, confirmando que os efeitos protectores da fucoxantina estão relacionados com via metabólica da AMPK [189]. ...
... In I Congreso de La Juventud Investigadora en Producción Primaria Sostenible y Seguridad y Calidad Alimentaria (JIPAS), 27-29/11/2019, in Lugo (Spain).As actividades biológicas e terapêuticas relacionadas com a fucoxantina têm sido repetidamente sublinhadas nas últimas décadas, em numerosos trabalhos científicos, que descrevem as suas capacidades antioxidante, anticancerígena, anti-hipertensiva, anti-inflamatória, antidiabética, antiobesidade, antiangiogénica e também fotoprotectora[39,47,50,65,68,[146][147][148][149][150], conforme descrito na Tabela 3. No entanto, até ao momento, a bioactividade mais estudada continua a ser a antioxidante, devido aos seus efeitos benéficos para a saúde e portanto a potenciais novas aplicações no sector alimentar e na indústria farmacêutica[144,151].Tabela 3. Resumo das bioactividades e efeitos da fucoxantina. Células de fibroblastos renais de macaco (Vero)[41] Inibição da acumulação de lipídios e da formação de ROS in vitro em adipócitos de rato 3T3-L1[156] Células de cancro de fígado humano Hep G2 pré-tratadas in vitro[156] ...
Thesis
Full-text available
In recent decades, numerous marine organisms have been shown to be a promising source of compounds of interest to the food industry, such as vitamins, minerals, polyunsaturated fatty acids, peptides, phenolic compounds, pigments, etc. Algae are among these organisms and have been used as food and traditional remedies, initially in Asian countries but are currently used all around the world. In addition to its good nutritional values, the presence of bioactive compounds has drawn the attention of different areas of research and several industries with the aim of promoting its application as a sustainable raw material for the obtention of new ingredients. Taking this into account, the general objective of this doctoral thesis was to explore the potential of macroalgae from the Galician coastline as a source of bioactives, which resulted as a final purpose to define the optimal conditions for different methodologies for the extraction of fucoxanthin from Undaria pinnatifida. In an initial screening stage, eight species of macroalgae were considered as possible sources of active compounds: Ulva rigida and Codium tomentosum from the Cholophyta group (green algae), Palmaria palmata and Porphyra purpurea from the Rodophyta group (red algae) and Himanthalia elongata, Laminaria ochroleuca, S. latissima and U. pinnatifida from the Ochrophyta group (brown algae), which are widely present on the Galician coastline and are currently used in the food industry. The chemical composition, nutritional analysis and antioxidant and anti-inflammatory properties of these species were analyzed, revealing a great variability between species and groups. However, the four species of brown algae showed a higher extraction yield, which is a fundamental parameter for the design of subsequent industrial processes. Based on this, brown algae were selected as the study group for future analyses. In a second stage, it was decided to assess the potential of algae group as a source of bioactive compounds. For this a few more species of brown algae were added to the ones previously used, whose use is not currently widespread, in order to increase the range of evaluation. The added species were Ascophyllum nodosum, Bifurcaria bifurcata, Fucus spiralis, Pelvetia canaliculata and Sargassum muticum, which are all species you can also find in the Galician coastline. In this study, the pigment composition and biological properties of these nine species of brown algae was carried out, using different solvents (ethanol, acetone, hexane, chloroform and ethyl acetate), in order to evaluate the suitability of each one and select the most appropriate. The pigment analysis showed the presence of a wide variety of pigments, highlighting fucoxanthin, which was found in large quantities in all studied species but specially in U. pinnatifida. This carotenoid has gained relevance for a few decades, due to its numerous biological properties, corroborated both in vitro and in vivo. In fact, it has been considered as a functional ingredient for the development of various nutraceutical products, so this molecule was selected as the target compound and the algae U. pinnatifida as the principal extraction matrix. Additionally, ethanol and acetone were able to obtain higher yields, and they are both suitable to be used in the food industry, so they were chosen as extraction solvents. Once the target compound, matrix, and extraction solvents had been selected, the next step was to design a rapid method HPLC-DAD to quantify fucoxanthin from a large number of samples, in a simple way. This method was used for the optimization stage of the fucoxanthin extraction methods. Firstly, two kinetic studies were carried out to compare the efficiency of both solvents in fucoxanthin extraction. Based on the results, the most efficient solvent for its extraction was ethanol, which is considered a green solvent, suitable for the development of respectable industrial processes with the environment. Next, the extraction of fucoxanthin from U. pinnatifida was also carried out using innovative extraction techniques as MAE and UAE. This, methodology was used to determine on a laboratory scale, the conditions that allowed the best fucoxanthin extraction performance based on the previously selected factors. In the optimization, variables like power, extraction time and solvent concentration were evaluated, using a response surface methodology. This procedure was used with two different technologies: MAE and UAE, to contrast its effectiveness, and they were compared with a conventional method using a standard SAE. The results showed that through UAE technology the obtained yield was much higher than the one obtained with conventional techniques and also the ones reported in literature. Lastly, once the best conditions for extraction were determined and the kinetic of fucoxanthin’s extraction was known, the results were discussed with an algae factory and a pilot plant was designed, according to their preferences and specifications, to obtain extracts rich in fucoxanthin at a larger scale. In the pilot plant designed, the alga is washed, desiccated and pulverized preparing it to the extraction in an industrial reactor. After the extraction, the content is filtered, obtaining an extract rich in fucoxanthin, which is finally dehydrated and stored. The final extract was later incorporated into a food product with added nutritional value.
... It was found that lipid oxidation induced by retinol deficiency (plasma and liver) was reduced by the supplementation of fucoxanthin (plasma 7-85% and liver 24-72%) versus β-carotene (plasma-51-76% and liver 33-65%) by enhancing the activity of catalase and glutathione transferase enzymes. Similarly, Jang et al. [116] reported the ability of fucoxanthin from Laminaria japonica to impart hepatoprotective effects under oxidative stress, suggesting its inclusion in the formulation of nutraceuticals. For other algae-derived carotenoids, protective properties such as cardioprotective, hepatoprotective, photoprotective, renal protective, and various health-promoting and beneficial properties such as antioxidant, anti-obesity, antitumor, antidiabetic, anti-inflammatory, and hepatoprotective have been confirmed in the literature [29,45,91,101,[115][116][117][118][119][120][121][122][123]. ...
... Similarly, Jang et al. [116] reported the ability of fucoxanthin from Laminaria japonica to impart hepatoprotective effects under oxidative stress, suggesting its inclusion in the formulation of nutraceuticals. For other algae-derived carotenoids, protective properties such as cardioprotective, hepatoprotective, photoprotective, renal protective, and various health-promoting and beneficial properties such as antioxidant, anti-obesity, antitumor, antidiabetic, anti-inflammatory, and hepatoprotective have been confirmed in the literature [29,45,91,101,[115][116][117][118][119][120][121][122][123]. Due to these properties, algae-derived carotenoids have been investigated for various applications. ...
Article
Full-text available
Recently, the isolation and identification of various biologically active secondary metabolites from algae have been of scientific interest, with particular attention paid to carotenoids, widely distributed in various photosynthetic organisms, including algal species. Carotenoids are among the most important natural pigments, with many health-promoting effects. Since the number of scientific studies on the presence and profile of carotenoids in algae has increased exponentially along with the interest in their potential commercial applications, this review aimed to provide an overview of the current knowledge (from 2015) on carotenoids detected in different algal species (12 microalgae, 21 green algae, 26 brown algae, and 43 red algae) to facilitate the comparison of the results of different studies. In addition to the presence, content, and identification of total and individual carotenoids in various algae, the method of their extraction and the main extraction parameters were also highlighted.
... Previous studies of the activity of fucoxanthin in U87MG glioblastoma cells have shown the modulation of several individual proteins involved in apoptosis, including B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, and caspase-9, all of which are involved in the PI3K/Akt pathway [30]. In addition, both in hepatocytes and in immortal human cell line (HeLa) cells, fucoxanthin has been shown to stimulate the adenosine monophosphate -activated protein kinase (AMPK) pathway to induce autophagy and cytoprotection [31]. In HeLa cells, fucoxanthin also inhibits Bcl-2, inducing Bax production and caspase-3 cleavage [32], while the deacetylated human metabolite of dietary fucoxanthin, fucoxanthinol (Fig 1D), modulates the NF-κB pathway, caspase activity, Bcl-2 proteins, MAPK, PI3K/Akt, JAK/STAT, activator protein 1 (AP-1), and growth arrest and DNA damage-inducible 45 (GADD45) [33]. ...
Article
Full-text available
Pathway analysis is an informative method for comparing and contrasting drug-induced gene expression in cellular systems. Here, we define the effects of the marine natural product fucoxanthin, separately and in combination with the prototypic phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002, on gene expression in a well-established human glio-blastoma cell system, U87MG. Under conditions which inhibit cell proliferation, LY-294002 and fucoxanthin modulate many pathways in common, including the retinoblastoma, DNA damage, DNA replication and cell cycle pathways. In sharp contrast, we see profound differences in the expression of genes characteristic of pathways such as apoptosis and lipid metabolism, contributing to the development of a differentiated and distinctive drug-induced gene expression signature for each compound. Furthermore, in combination, fucoxanthin synergizes with LY-294002 in inhibiting the growth of U87MG cells, suggesting complemen-tarity in their molecular modes of action and pointing to further treatment combinations. The synergy we observe between the dietary nutraceutical fucoxanthin and the synthetic chemical LY-294002 in producing growth arrest in glioblastoma, illustrates the potential of nutri-pharmaceutical combinations in targeting this challenging disease.
... 28 Meanwhile, producers extract fucoidans from brown algae, with l-fucose as their main monomer, known for their antiproliferative, antiangiogenic, and anticancer properties. 29,30 The food industry relies on carrageenans from red algae and linear sulfated polysaccharides to modify food textures, notably in fermented products like "tofu". 31 Lastly, agar, also from red algae, plays a crucial role in the gelatin and confectionery industries due to its versatile applications. ...
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Dietary supplements play a crucial role globally in reducing the incidence of emerging diseases. Over the last decade, there has been substantial exponential growth in their industrial production, ensuring greater accessibility to a more significant number of individuals. Marine-derived nutraceuticals represent around 50% of the market, encompassing numerous bioactive compounds and secondary metabolites obtained from marine organisms, especially seaweeds, fish, and marine bacteria. They are characterized by their antimicrobial, anti-inflammatory, antioxidant, and anticancer activities through different mechanisms of action, such as the activation of enzymatic factors and transcription factors that modulate various physiological pathways. This review highlights the importance of the main bioactive compounds from marine organisms and describes recent advances in their beneficial role in health.
... The anti-oxidant activity of fucoxanthin is closely related to the Nrf2 and AMPK signaling pathway. Fucoxanthin activates the Nrf2 and AMPK signaling pathway to reduce oxidative stress [109,110]. Activation of the AMPK pathway in the liver has been shown to improve the mitochondria's ability to resist oxidative damage. Meanwhile, the antifibrogenic activity of fucoxanthin is mainly through the inhibition of TGF-β1. ...
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... В медицине препараты на основе слоевищ ламинарии широко используются для профилактики диабета [8] и йододефицитных состояний [9,10], а также при других заболеваниях, в частности для лечения запоров [11][12][13]. Широко изучаются антиоксидантные [14] и гепатопротекторные [14,15] свойства этих растений. ...
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Epidemiological investigations have shown that overcoming the risk of cancer is related to the consumption of green vegetables and fruits. Many compounds from different origins, such as terrestrial plants and marine and microbial sources, have been reported to have therapeutic effects of which marine sources are the most important because the diversity of marine life is more varied than other sources. Fucoxanthin is one important compound with a marine origin and belongs to the group of carotenoids; it can be found in marine brown seaweeds, macroalgae, and diatoms, all of which have remarkable biological properties. Numerous studies have shown that fucoxanthin has considerable medicinal potential and promising applications in human health. In this review, we summarize the anticancer effects of fucoxanthin through several different mechanisms including anti-proliferation, induction of apoptosis, cell cycle arrest and anti-angiogenesis, and its possible role in the treatment of cancer.
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