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Effective Inhibition of Skin Cancer, Tyrosinase, and Antioxidative
Properties by Astaxanthin and Astaxanthin Esters from the Green
Alga Haematococcus pluvialis
Ambati Ranga Rao,
†,§
H. N. Sindhuja,
‡
Shylaja M. Dharmesh,*
,‡
Kadimi Udaya Sankar,
#
Ravi Sarada,
†
and Gokare Aswathanarayana Ravishankar
†,⊥
†
Plant Cell Biotechnology Department,
‡
Biochemistry & Nutrition Department, and
#
Food Engineering Department, Central Food
Technological Research Institute, CSIR, Mysore 570 020, Karnataka, India
§
Institute of Ocean and Earth Sciences, University of Malaya, Kuala Lumpur 50603, Malaysia
⊥
Dayananda Sagar Institutions, Dr. C. D. Sagar Center for Life Sciences, Shavige Malleshwara Hills, 5th Floor, Kumaraswamy Layout,
Bangalore 560 078, India
ABSTRACT: Astaxanthin mono- (AXME) and diesters (AXDE) were characterized and examined for anticancer potency with
total carotenoids (TC) and astaxanthin (AX) against UV−7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer model
in rat. At 200 μg/kg bw, AXDE and AXME reduced UV-DMBA-induced tumor incidences up to 96 and 88%, respectively, when
compared to AX (66%) and TC (85%). UV-DMBA has been known to generate high levels of free radicals and tyrosinase
enzyme, leading to characteristic symptoms of skin pigmentation and tumor initiation. Intriguingly, ∼7-fold increase in tyrosinase
and 10-fold decrease in antioxidant levels were normalized by AXDE and AXME as opposed to only ∼1.4−2.2-fold by AX and
TC, respectively. This result together with the appearance of 72 and 58 ng/mL of retinol in the serum of respective AXE-treated
(AXDE + AXME) and AX-treated animals suggested that better anticancer potency of AXEs could be due to increased
bioavailability.
KEYWORDS: microalgae, AX, AXME, AXDE, UV-DMBA, skin cancer, retinol
■INTRODUCTION
Developing novel strategies to prevent skin cancer represents a
desirable goal due to the rise in the incidence of skin cancer
patients throughout the world.
1
According to the World Cancer
Report, skin cancer constitutes ∼30% of all newly diagnosed
cancers in the world.
2
This rise in incidence has been attributed
to overexposure of skin to sun/UV light, due to reduction in
the ozone in the atmosphere.
3
Skin cancer thus is a disease in which malignant cells are
found in the outer layer of the skin. Melanoma is one of the
most serious consequences of skin cancer where melanocytes
proliferate actively with enhanced accumulation of melanin
pigment leading to pigmentation and discoloration of the skin
in addition to tumor formation. Up-regulated levels of
tyrosinase enzyme appear to contribute significantly to the
enhanced synthesis and accumulation of melanin in melano-
cytes.
Like most cancers, melanoma is best treated when it is
diagnosed early. Melanoma can metastasize quickly to other
parts of the body through the lymph system or through the
blood. Most of the cytotoxic drugs used presently in cancer
therapy are highly toxic to a wide spectrum of tissues such as
the gastrointestinal tract, bone marrow, heart, lungs, kidney,
and brain. Latrogenic failure of these organs has been observed
frequently as a cause of death from cancer.
4
Melanomas are
difficult to eradicate by chemotherapy because they exhibit a
well-known phenomenon, “chemoresistance”. Expression of
survivin molecules in the cells appears to cause drug resistance,
resulting in very little option for curing the disease. Attempts
are underway with the use of tyrosinase inhibitors,
5
particularly
from natural sources, to overcome chemoresistance and to
avoid side effects.
6
Indeed, much progress is being made in the
direction of pharmacological evaluation of various plant
products and dietary sources with the hope of achieving
effective chemoprevention.
7
Extensive research has been done on Haematococcus pluvialis,
a unicellular green alga, in our laboratory including its
biotechnological production, characterization of type of
astaxanthin (AX), astaxanthin esters (AXEs), etc. Astaxanthin
esters are unique, constituted by 70% of monoesters, 15−20%
of diesters, and 4−5% of free forms, indicating the
predominance of esterified astaxanthin forms in H. pluvialis as
opposed to free forms in other plant sources.
8
Antioxidant
activities 100 and 10 times greater than those of vitamin E and
β-carotene have been reported in AX.
9,10
Recently developed
downstream processing for the large-scale production of AX
and AXEs may potentiate their uses as anticancer alternatives.
11
Furthermore, intriguing studies by Camera et al.
12
and Savouré
et al.
13
implied that different carotenoids exhibit varied
potential to offer protection against UV-induced skin cancer.
Among the three important carotenoids, AX, canthaxanthin
(CX), and β-carotene (BC), AX, which is an oxocarotenoid, has
a superior preventive effect toward photo-oxidative changes in
Received: October 30, 2012
Revised: March 10, 2013
Accepted: March 10, 2013
Published: March 11, 2013
Article
pubs.acs.org/JAFC
© 2013 American Chemical Society 3842 dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−3851
cell culture. Results of the present investigation add to the
previous observation that AXEs are more protective than AX in
UV−7,12-dimethylbenz(a)anthracene (DMBA)-induced skin
cancer in rats. Studies also address two possible mechanisms
of increased potency of AXEs, such as free radical scavenging
activity per se, as well as vitamin A activity in terms of observing
the generation of relative levels of retinol from AX and AXEs.
The data revealed for the first time that AXEs are more potent
than AX and have an impact on their uses as anticancer
alternatives, because they are available in great abundance in H.
pluvialis with the defined protocol for their optimization.
■MATERIALS AND METHODS
H. pluvialis.H. pluvialis was obtained from Sammlung von
Algenkulturen, Pflanzenphysiologisches Institute, Universitat Gottin-
gen, Gottingen, Germany, and was maintained on modified
autotrophic bold basal medium and agar slants.
8
Extraction, Isolation, and Characterization of AX and AXEs.
Total carotenoid (TC) from H. pluvialis biomass was extracted and
characterized as described previously.
14,15
Briefly, TC was subjected to
preparative thin layer chromatography (TLC) using the solvent system
acetone/hexane (3:7, v/v) and separated astaxanthin (AX),
astaxanthin monoester (AXME), and astaxanthin diester (AXDE)
bands from TLC plates and characterized by mass spectra.
HPLC Analysis of AX, AXEs, and Retinol. Isolated AX and AXEs
from H. pluvialis and retinol in serum and liver were analyzed using a
HPLC (Shimadzu 10AS, Kyoto, Japan) reverse phase 25 cm ×4.6
mm, 5 μm, C18 column (Wakosil 11 5C 18RS) with an isocratic
solvent system consisting of dichloromethane/acetonitrile/methanol
(20:70:10, v/v/v) at a flow rate of 1.0 mL/min.
16,17
AX, AXEs, and
retinol were monitored at 476 and 325 nm with a UV−visible detector
(Shimadzu). Peak identification and λmax values of these components
were confirmed by their retention times and characteristic spectra of
standard chromatograms recorded with a Shimadzu model LC-10AVP
series equipped with an SPD-10AVP photodiode array detector. They
were quantified from their peak areas in relation to the respective
reference standards.
Characterization of AX and AXEs by Liquid Chromatog-
raphy−Mass Spectroscopy (LC-MS) in Atmospheric Pressure
Chemical Ionization (APCI). Isolated AX and AXEs from H. pluvialis
biomass were characterized by using the Waters 2996 modular HPLC
system (autosampler, gradient pump, thermoregulator, and DAD),
coupled to a Q-Tof Ultima (UK) mass spectrometer. In brief, the
APCI source was heated at 130 °C, and the probe was kept at 500 °C.
The corona (5 kV), HV lens (0.5 kV), and cone (30 V) voltages were
optimized. Nitrogen was used as sheath and drying gas at 100 and 300
L/h, respectively. The spectrometer was calibrated in the positive
mode, and [M + H]+ions were recorded. Mass spectra of AX and
AXEs were acquired with m/z400−2000 scan range.
Effect of AX and AXEs on UV-DMBA-Induced Skin Carcino-
genesis in Vivo. Healthy albino Wistar rats (220 ±5 g) used for the
experiments were maintained under standard conditions of temper-
ature, humidity, and light and were provided with standard rodent
pellet diet (M/s. Sai Durga Feeds, Bangalore, India) and tap water ad
libitum. Animals for the study were approved by the Institutional
Animal Ethical Committee (IAEC No. 116/08), which follows the
guidelines of the CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments on Animals, reg. no. 49, 1999),
government of India, New Delhi, India. All animals were divided into
14 groups (n= 6 for each group), their body weights were recorded,
and their backs were shaved prior to the start of experiment. TC, AX,
and AXEs dissolved in ground nut oil were intubated to groups 3, 4, 7,
10, and 13 at 100 μg/kg body weight (bw) and groups 6, 9, and 12 at
200 μg/kg bw, respectively. Group I is the healthy group, whereas
group II animals were exposed to UV and DMBA and hence served as
the “cancer-induced”group. Groups 5, 8, 11, and 14 served as sample
control groups and were treated with only TC, AX, AXME, and AXDE
at μg/kg bw, respectively. The samples/standard was intubated prior
to cancer induction for 14 days. From the 15th day onward UV
radiation (West Berlin, Universal-UV-Lamp, 254 nm, 200 V ∼50 Hz)
daily (30 min/day) followed by DMBA (100 μg in 100 μL of acetone/
rat, weekly twice applied on skin) was given. Samples were given
everyday throughout the experimental period, which was about 60
days.
Measurement of Tumor Index. Tumors and skin lesions were
detected in UV-DMBA-treated rats. The tumor index was calculated as
described by Koul et al.
18
Tumor volume and burden were calculated
using the following formulas: mean tumor volume = 4/3πr3(r= mean
tumor radius in mm); mean tumor burden = mean tumor volume ×
mean number of tumors. The intensity of tumor was also calculated by
histopathological analysis using Image J software.
19
Assay for Tyrosinase, Protein, and Antioxidant Enzymes in
the Skin Homogenate and Serum. Tyrosinase enzyme activity was
measured in serum and skin homogenate in control and UV-DMBA-
treated groups following the standardized protocols employed
earlier.
20,21
Tyrosinase enzyme activity was measured in the serum
and skin homogenates of all groups of animals using L-Dopa as
substrate with slight modifications. L-Dopa (0.1 mL of a 1 mg/mL
solution) was mixed with 0.8 mL of 0.1 M phosphate buffer (pH 6.0)
and incubated with 0.1 mL of serum/skin homogenates at 37 °C for
15 min. Dopachrome formation was measured fluorometircally
(excitation, 360 nm; and emission peak, 720 nm). The increased
tyrosinase activity was determined by the increase in the absorbance at
excitation of 360 nm and emission of 720 nm. The protective ability of
treated groups with astaxanthin and its esters on the tyrosinase activity
was determined and quantitated.
17
Protein content was determined using the method described.
22
Superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)
levels and TBARS were measured as per the protocol described earlier
by our group.
23−26
Hematological and Histopathological Analysis. EDTA anti-
coagulated blood samples were used to obtain a complete blood count
with a Hemavet Mascot Multispecies Hematology System Counter
1500R (Ravi Diagnostic Laboratory, Mysore, Karnataka, India).
Lymphocytes, mean cell hemoglobin count, platelets, mean cell
volume, packed cell volume, red blood cells, mean cell hemoglobin,
hemoglobin, and neutrophils were analyzed. For histopathological
studies, skin tissue samples were fixed in 10% buffered formalin for 24
h. The processed tissues were embedded in paraffin blocks, and
sections made were stained with hematoxylin and eosin dye. The
sections were analyzed by observation under light microscope (Leitz,
Germany) at 10×magnification. Tumor areas in untreated and
sample-treated sections were localized using Image J software. Results
were compared between the groups using SPSS Statistics 17.0. OA
one-way ANOVA test followed by a post hoc Tukey test was
performed.
Determination of Bioavailability of AX and AXEs in Different
Animal Groups. AX is known to convert into retinol. Hence, to
determine whether the observed bioactive potential of AX and AXEs
in prevention of UV-DMBA-induced rats is due to the generation of
retinol, AX and retinol were measured in the serum and liver by HPLC
using our earlier procedures.
16,17
Toxicological Studies. Activities of the enzymes serum glutamate
oxaloacetate transaminase (SGOT), serum alkaline phosphatase
(SALP), and serum glutamate pyruvate transaminase (SGPT) in
serum and skin were estimated in healthy control, sample control, and
UV-DMBA-treated groups using standard enzyme kits.
27−29
Statistical Analysis. Results were expressed the mean ±standard
deviation (SD) of six. The data were analyzed by ANOVA using
Microsoft Excel XP (Microsoft Corp., Redmond, WA, USA), and the
post hoc mean separations were performed by Duncan’s multiple-
range test at p< 0.05.
■RESULTS AND DISCUSSION
Characterization of AX and AXEs by HPLC and LC-MS
(APCI). The total carotenoid (TC) constituted about 2−3% of
H. pluvialis total biomass. Of TC, AX and its esters (AXME +
AXDE) constituted ∼2 and 80%, respectively. They could be
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513843
separated from total carotenoid extract of H. pluvialis with
acetone/hexane (3:7, v/v) mobile phase on silica-based thin
layer chromatography with different Rfvalues. AX had an Rfof
∼0.54, whereas astaxanthin monoesters (AXME, Rf= 0.77) and
diesters (AXDE, Rf= 0.82) showed characteristic mobility. AX
and AXEs were identified by absorption spectra at 470−476 nm
by HPLC, and both were obtained in 98% purity. Because there
was a good resolution between AX, AXME, and AXDE on
TLC, respective spots were scraped and reconfirmed their
mobility with the same chromatographic system, and such pure
components isolated by preparative thin layer chromatography
were identified as AX and AXEs by LC-MS. LC-MS (APCI)
positive mode and the MS data used for the identification of
AX, AXME, and AXDE are summarized in Table 1. The mass
spectrum was obtained from an AXME (ME C16:0,MEC
17:2,
ME C17:1,MEC
17:0,MEC
18:4,MEC
18:3,MEC
18:2,MEC
18:1).
Because only mass differences between quasimolecular and
fragment ions were used for assignment of acyl chains, the
location of double bonds could not be determined by the mass
spectrum. Thus, many isomers of astaxanthin esters in H.
pluvialis could not be identified unequivocally. We have
observed that the fragmentation pattern of astaxanthin esters
was dominated by the loss of fatty acid and water. Protonated
[M + H]+resulted from the positive ion mode. A total of eight
AXME have been identified. The mass spectrum was obtained
from an AXDE (DE C16:0/C16:0,DEC
16:0/C18:2,DEC
18:1/C18:3,
DE C18:1/C18:2,DEC
18:1/C18:1)inH. pluvialis. The basic peaks
of other astaxanthin diesters showed characteristic fragment
ions of losing one fatty acid, but their fragment ions of losing
the second fatty acid had relatively weaker intensity.
Effect of TC, AX, and AXEs on UV-DMBA-Induced Skin
Carcinogenesis in in Vivo Model. Having identified
uniquely modified carotenoids such as esterified AX in a
natural source, H. pluvialis, and taking the fact that carotenoids
exhibit anticancer property differentially, through different
mechanisms, the following were hypothesized in this study: (1)
the comparative anticancer efficacy of differentially esterified
AX (AX, AXME, and AXDE) in UV-DMBA-induced skin
cancer model; (2) the probable mechanism of action such as
free radical scavenging or vitamin A activity; and (3) differences
in the ability of AX and AXEs on retinol and TBARS in the
plasma of experimental animals. These questions were
addressed because AX has been reported to exhibit multiple
potencies of protecting skin during the physiological conditions
of growth and development, when they are exposed to photo-
oxidation. Also, evidence has been accumulated in the literature
regarding the accumulation of carotenoids in the epidermal
cells of skin, attributing the skin protective abilities. The aim of
the current work indeed is to understand the role of AXEs
under the above-mentioned conditions. Accordingly, experi-
ments were designed with the purified fractions of AX and
AXEs from H. pluvialis with the standardized protocol of our
laboratory against the skin during UV exposure condition that
is known to cause carcinogenic conditions in humans. Because
UV is known to induce reactive oxygen species (ROS), the
ability of these AX and AXEs to inhibit TBARS and the
subsequent induction of tyrosinase that happens due to
depletion of antioxidant GSH and antioxidant enzymes that
are known to defend the animals against oxy radicals were
studied. Retinol content was also measured in both the serum
and liver of animals, which indicates the efficiency of conversion
of AX and AXEs toward vitamin A activity. Results were
compiled with appropriate statistical methods and interpreted
to arrive at the role of AX and AXEs against skin cancer.
Exposure of UV-DMBA to rats showed alterations in the
texture of the skin, in addition to inflammatory patches. Skin
lesions with tumor nodules/tumor mass, angiogenesis, and
inflammation was observed. No such skin tumor lesions and
bleeding are noted in either healthy or sample control groups
(Figure 1A,C,E,G,I). Animals pretreated with TC, AX, and
AXEs showed different levels of tumor reduction (Figure
1B,D,F,H,J). AXEs, however, showed the best inhibition with
88−96% reduction in tumor index at 200 μg/kg bw (Table 2).
AXDE was found to be better than AXME (1.1-fold) among
AXEs. The differential effects of TC, AX, AXME, and AXDE
appear to be due to their structural variations.
Quantitation of the data presented in Table 2 indicated that
all fractions showed good protection; however, maximum
protection was observed with AXDE followed by AXME, TC,
and AX. It is interesting to also compare our data with those
Choi et al.;
30
the efficacy of AX in humans would be better by
2.5-fold because the area under curve (AUC) of AX after its
oral administration at a dose of 40 mg in human subjects, 80.8
μg min/mL, was close to 77.3 μg min/mL after its oral
administration at a dose of 100 mg/kg in rats. In other words, it
is possible to observe a 2.5-fold better efficacy of AX in offering
anticancer property. However, because AXEs appear to be
more potent, it would be interesting to compare their
pharmacokinetics to understand their practical feasibility.
30
Furthermore, it is also important to highlight here the
significance of the slight modification in the methodology of
UV-DMBA-induced cancer in rats. Conventionally, significant
levels of tumors were induced only after 20−30 weeks
31−33
as
opposed to 60 days in the current study, where we could
observe the same between 8 and 9 weeks. This early induction
of tumor could be due to exposure to UV radiation daily (30
min/day) and DMBA (weekly twice). The current study thus
highlights the mode of early induction of skin tumors in an
experimental animal model, which offers a desirable reduction
of time in animal experiments.
Histopathological Changes. Histopathological observa-
tions revealed that skin lesions with normal histological features
were observed for healthy controls (Figure 2A). Animals fed
with just samples alone also showed normal structures (Figure
Table 1. LC-MS (APCI) Data for Astaxanthin and
Astaxanthin Esters
m/z
no. [M] [M + 2H −FA1]+1 MS2compound
a
1597 [M −H2O] 579 free astaxanthin
2835 579 836 ME C16:0
3845 579 846 ME C17:2
4847 579 848 ME C17:1
5849 579 850 ME C17:0
6855 579 854 ME C18:4
7857 579 856 ME C18:3
8859 579 858 ME C18:2
9861 579 859 ME C18:1
10 1072 579 1071 DE C16:0/C16:0
11 1096 579 1095 DE C16:0/C18:2
12 1120 579 1119 DE C18:1/C18:3
13 1122 579 1121 DE C18:1/C18:2
14 1124 579 1123 DE C18:1/C18:1
a
ME, monoesters; DE, diesters.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513844
2C,E,G,I). UV-DMBA-induced rats showed greater changes in
the epidermis and dermis including irregular distribution with
finger-like papilloma indicative of cancerous growth (Figure
2B). The tumors were composed of focal proliferation of
squamous cells and characterized by the presence of some
necrotic cells, keratinization, and epithelial pearls. AXDE-
treated groups showed reduction in these lesions, although
marginal epidermal thickness relative to that of the normal
group was observed (Figure 2J). Only partial protection as per
skin lesions was observed in AXME- and TC-treated groups,
suggesting that esters can offer better protection (Figure 2F,H)
than AX (Figure 2D). Tumor areas in control and treated
sample sections were localized using Image J software
16
(Figure
3). Results were compared between the groups using SPSS
Statistics 17.0. A one-way ANOVA test followed by a post hoc
Tukey test was performed.
Figure 1. Photographs of rat skin tumors: (control groups) healthy
(A), AX200*(C), TC200*(E), AXME200*(G), and AXDE200*(I);
(UV-DBMA-treated groups) UV-DMBA (B), UV+AX200*(D), UV
+TC200*(F), UV+AXME200*(H), and UV+AXDE200*(J). *,μg/
kg bw.
Table 2. Quantitative Differences in Tumor Incidences in
Healthy, UV-DMBA, and UV-DMBA+AX/AXE Samples
a
group
tumor
incidence
(%) mean tumor
burden (mm3)reduction in mean
tumor burden (%)
healthy c
UV-DMBA 100 a 748.84 ±89.13 a 0
AX200 c −
b
−
b
−
b
TC200 c −
b
−
b
−
b
AXME200 c −
b
−
b
−
b
AXDE200 c −
b
−
b
−
b
AX200*44.17 b 258.34 ±38.47 b 65.51 c
TC200*19.8 c 115.73 ±12.70 c 84.54 b
AXME200*15.85 d 87.38 ±9.85 d 88.33 b
AXDE200*6.96 e 24.34 ±4.33 e 96.74 a
a
Each value represents the mean ±SD (n= 6). Values not sharing a
similar letter within the same column are significantly different (p<
0.05) as determined by ANOVA. *,μg/kg bw. AX, astaxanthin; TC,
total carotenoid; AXME, monoester of astaxanthin; AXDE, diester of
astaxanthin from H. pluvialis.
b
−, not found.
Figure 2. Histopathological control groups: healthy (A), AX200*(C),
TC200*(E), AXME200*(G), and AXDE200*(I). UV-DBMA
+samples-treated groups: UV-DMBA (B), UV+AX200*(D), UV
+TC100*(F), UV+AXME200*(H), and UV+AXDE200*(J) showed
a significant epidermal thickness in the sections of UV-DMBA-induced
group when compared to healthy and sample control groups. *,μg/kg
bw.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513845
Hematological Changes. Hematological changes were
observed in UV-DMBA-treated groups when compared to
healthy control and sample control groups. Hematological
parameters such as lymphocytes, mean cell hemoglobin count,
platelets, mean cell volume, packed cell volume, red blood cells,
mean cell hemoglobin, hemoglobin, and neutrophils were
observed. Platelets, lymphocytes ,and neutrophil counts were
affected significantly (Table 3).
Astaxanthin and Retinol Levels in Serum and Liver.
Vitamin A is essential for a number of physiological processes,
such as regulation of cell differentiation, cell proliferation,
vision, and reproduction. Because carotenoids are the major
sources of vitamin A/retinol, their levels were measured in the
serum and liver of all groups of animals (Figure 4). The
maximum levels of astaxanthin (366 ng/mL) and retinol (72
ng/mL) were found in the serum of the AXDE-treated group,
followed by AXME, when compared to healthy control and
other groups (Figure 4A,B). In the liver, AXDE/AX-treated
groups contained 450/400 and 18/6 ng/g of liver tissue of
astaxanthin and retinol, respectively, suggesting that biocon-
version to retinol is much better with AXDE than with AX
alone (Figure 4C,D). Furthermore, it is interesting to note that
maximum depletion of AX and retinol was observed in the
AXDE-treated group followed by UV-DMBA-exposed animals
relative to AXDE control, indicating that the utilization of
retinol is greater during the UV-DMBA-induced condition. The
data further emphasize that AXDE may offer protection to
animals against UV-DMBA via its conversion to retinol. Similar
results were observed in the liver also.
Inhibitory Effect of Tyrosinase Activity. The rate-
limiting enzyme tyrosinase is responsible for melanin pigment
biosynthesis in human skin, which takes place within specialized
organelles known as melanosomes. Skin tyrosinase has been
widely used as the target enzyme for screening and character-
izing potential tyrosinase inhibitors.
34
The activity was found to
be increased by 7.4-fold in UV-DMBA-treated groups, and this
was inhibited up to 4.5- and 3.0-fold in AXDE- and AXME-
treated groups, respectively (Table 4).
Changes in Antioxidant Enzymes and Lipid Perox-
idation Levels in Serum and Skin Homogenates. Effects
of TC, AX, AXME, and AXDE on the antioxidant enzymes and
TBARS levels were measured in all groups of experimental
animals. Table 5 indicates the changes in the antioxidant
enzymes and lipid peroxidation levels in the serum of UV-
DMBA-induced rats. SOD levels increased by ∼2-fold in the
serum, and CAT and GSH levels were found to be depleted by
1-fold. Furthermore, an approximately 10-fold increase in
TBARS levels in UV-DMBA-treated groups observed was
normalized up to 65% upon treatment with AXEs. The
antioxidant enzymes and lipid peroxidation levels were also
measured in skin homogenate (Table 5). The SOD level
increased in skin by 2.7-fold. CAT and GSH levels decreased by
1-fold in the UV-DMBA-treated group, which was restored to
normal levels upon treatment with AX and AXEs. A 10.6-fold
increase in TBARS in UV-DMBA-treated skin tissues was
further recovered up to 60% upon treatment.
SGPT, SGOT, and SALP Levels in Serum and Skin
Homogenates. SGPT, SGOT, and SALP levels were
measured in serum and skin homogenates of UV-DMBA-
induced rats (Table 6). The data revealed the normalization of
these enzymes by treated samples. Toxicity profiles were also
studied for AX, TC, AXME, and AXDE. SGPT, SGOT, and
SALP showed enhancement of activities in serum and skin
homogenates of UV-DMBA-treated groups. SGPT (1.8-fold),
SGOT (1.7-fold), and SALP (1.8-fold) increased in the serum
of the UV-DMBA-induced group, whereas the enzyme activities
were modulated significantly in the AXDE-treated group. In the
case of skin homogenates, 1.9-, 1.6-, and 2.1-fold increases in
SGPT, SGOT, and SALP activities, respectively, were observed
in UV-DMBA-induced groups. However, treatment with AXEs
resulted in maximum recovery.
Tyrosinase Inhibitory Potentials of AX and AXEs in
Vitro. It was found that AXDE and AXME had shown potent
inhibitory effects on L-Dopa oxidase activity of tyrosinase and
that the inhibitory activities increased with concentrations.
Figure 3. Analysis of tumor intensity: tumor intensity was quantitated
with biometric analysis of histopathological sections of AX-, TC-,
AXME-, and AXDE-treated groups. Results were compared with
healthy, UV+DMBA, and sample controls. Data showed significant
reduction in the tumor intensity in terms of tumor area as obtained by
Image J analysis in treated groups when compared to that of only UV-
DMBA-exposed group of animals. One-way ANOVA followed by post
hoc Tukey test indicated the level of significance, where pvalues are
indicated by ∗∗∗ p< 0.001. *μg/kg bw.
Table 3. Hematological Analysis of Healthy, UV-DMBA, and UV-DMBA+AX/AXE Samples
a
group PLT (×103/μL) LYM (%) LYM#(×103/μL) neutrophils
healthy c 904 ±6.00 b 76.6 ±3.01 d 7.60 ±1.23 d 19.34 ±1.78 b
UV+DMBA 759 ±6.31 e 86.9 ±5.10 a 13.9 ±1.36 a 10.45 ±1.40 d
AX200*993 ±8.90 a 71.8 ±6.72 c 6.43 ±0.92 e 23.75 ±1.85 a
TC200*833 ±6.62 d 81.3 ±4.61 b 8.30 ±1.04 c 14.45 ±2.08 c
AXME 200*868 ±7.73 c 81.9 ±3.40 b 9.10 ±1.37 b 11.23 ±1.82 d
AXDE 200*792 ±6.08 f 76.1 ±3.82 d 8.2 ±0.70 c 19.83 ±2.31 b
a
Each value represents the mean ±SD (n= 6). Values not sharing a similar letter within the same column are significantly different (p< 0.05) as
determined by ANOVA. *,μg/kg bw. PLT, platelet count; LYM, lymphocytes; AX, astaxanthin; TC, total carotenoid; AXME, monoester of
astaxanthin; AXDE, diester of astaxanthin from H. pluvialis.
Journal of Agricultural and Food Chemistry Article
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AXDE had the highest tyrosinase inhibitory activity with an
IC50 of 2.12 μg/mL followed by AXME (IC50 = 3.5 μg/mL)
and was found to be 2.4−2.8-fold better than AX and TC,
respectively (Figure 5).
Toxicity Studies. Body weight and relative organ weight at
the termination of the experiment have been observed. There is
no significant difference in the body weight gain profile, and the
corresponding values of low and high doses were comparable
with those of the control. The oral administration of TC, AX,
AXME, and AXDE did not cause any apparent changes in
clinical signs such as survivability or any gross visible changes
attributable to toxicity in the organ weights of rat. There was no
significant difference either in the biochemical profile or in the
serum or skin homogenate of various groups of animals or in
behavioral aspects. Similar results were observed when
astaxanthin-rich H. pluvialis biomass was fed to rats and no
adverse effects on animals were found.
10
Melanoma is a relatively common and one of the most
malignant tumors in humans. The social impact of skin cancer
leading to melanoma is significant because it has a very poor
prognosis;
35
more importantly, many melanoma cases occur in
young individuals, and there is little effective treatment available
once it becomes metastatic.
36
During the transformation of
normal melanocytes into malignant ones, several steps of
reactions, including imbalance in the regulation of proliferation,
apoptosis, galectin-3 expression, and the enhanced activation of
tyrosinase enzyme, are key factors, and hence they can be used
as promising targets for management of the disease.
37
In the
current study, we examined the role of carotenoids from H.
pluvialis, a microalga which produces varieties of carotenoids,
particularly the presence of ∼80% of mono- and diesterified
forms of astaxanthin of total carotenoids as opposed to either
free astaxanthin in other plant sources. It is of current interest
to find out the bioactive potency of esterified astaxanthin in
comparison with astaxanthin alone. Differences in bioavail-
ability and bioconversion to vitamin A in comparison with the
standard astaxanthin are also of interest because astaxanthin
primarily functions as a precursor of vitamin A and vitamin A
exhibits anticancer property. UV-DMBA-induced skin cancer
model has been employed during the study. Histopathological,
biochemical, and hematological parameters have been analyzed
to determine either the diagnostic or prognostic value of AX
and AXEs.
The results of the study indicated for the first time that AXE
exhibited a 3-fold higher potential in inhibiting skin cancer
incidences, and the efficacy could be attributed to increased
bioavailability as revealed by higher levels of vitamin A as
retinol in the serum of AXE-treated animals than in those
animals ingesting AX or TC alone. Results were substantiated
by histopathological studies, where 2−3-fold increased
reduction of papillomas was observed in groups of animals
treated with AXEs when compared to AX-treated animals.
Furthermore, a normal histological pattern in the skin of
control as well as AX- and AXE-receiving rats indicated no
adverse effect on the skin. In UV-DMBA-treated rats, all of the
Figure 4. Astaxanthin and retinol content in the serum (A, B) and liver (C, D) of UV-DMBA-induced and sample-treated groups: UV-DMBA (1),
AX100*(2), AX200*(3), AX200*control (4), TC100*(5), TC200*(6), TC200*control (7), AXME100*(8), AXME200*(9), AXME200*
control (10), AXDE100*(11), AXDE200*(12), and AXDE200*control (13). Each value represents the mean ±SD (n= 6) of analyses. Values not
sharing a similar letter between the groups are significantly different (p< 0.05) as determined by one-way ANOVA. *,μg/kg bw.
Table 4. Effect of AX and AXE on Tyrosinase Activity in
Serum and Skin Homogenates of UV-DMBA-Induced
Experimental Rats
a
group serum (μmol/mg protein) skin (μmol/mg protein)
healthy c 0.19 ±0.07 i 16.5 ±0.40 g
UV+DMBA 21.33 ±3.07 a 119.33 ±9.04 a
AX100*13.21 ±3.14 b 72.27 ±2.70 b
AX200*11.03 ±1.72 c 60.06 ±3.40 d
AX200*c 0.32 ±0.07 h 1.03 ±0.14 h
TC100*11.46 ±2.14 c 78.57 ±9.44 b
TC200*9.18 ±0.65 e 52.62 ±7.45 c
TC200*c 0.30 ±0.16 h 1.07 ±0.85 h
AXME100*10.28 ±2.22 d 50.04 ±8.31 c
AXME200*8.24 ±0.70 f 39.27 ±6.73 e
AXME200*c 0.19 ±0.08 i 1.25 ±0.64 h
AXDE100*9.94 ±1.84 e 33.19 ±0.50 e
AXDE200*7.60 ±1.26 g 26.06 ±1.02 f
AXDE200*c 0.24 ±0.11 h 1.169 ±0.04 h
a
Each value represents the mean ±SD (n= 6). Values not sharing a
similar letter within the same column are significantly different (p<
0.05) as determined by ANOVA. *,μg/kg bw. C, control group; AX,
astaxanthin; TC, total carotenoid; AXME, monoester of astaxanthin;
AXDE, diester of astaxanthin from H. pluvialis.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513847
tumors were confirmed to be papillomas, whereas the extent of
hyperchromatia in the tumors of the rats that received AXEs
was observed to be approximately 2−3-fold less than that of the
TC/AX treatment. AXDE showed better potency than AXME
and AX. Results thus indicate the chemopreventive role of
AXEs in the potential management of skin cancer.
In addition, as it is known that AX exerts a beneficial effect
against several disorders by antioxidative properties, the current
results of anticancer potentials of skin cancer were correlated to
antioxidant capacities of AX and AXEs, which was established
from our laboratory previously. Results, however, reveal that
the protective effect is not directly proportional to the
antioxidant capacities because AXEs showed lowered antiox-
idant activity relative to AX and TC.
15
The data thus may open
up the possibility that metabolites released into the serum from
AX- and AXE-treated group may be more antioxidative in
nature or may be going through a nonantioxidative route such
as tyrosinase inhibition. This interpretation may be supported
by one of our previous studies that had revealed that photo-
oxidized lutein has more melanoma cell killing effect than the
lutein per se.
38
Also, observed results of the efficacy of AXDE <
AXME < AX are also supported by increased inhibition of lipid
peroxidation and enhancement of antioxidant and antioxidant
enzyme levels in AXDE-treated groups followed by AXME- and
AX-treated groups. Lipid peroxidation has been reported to
play an important role in the control of cell proliferation and to
induce cytotoxicity and cell death
39,40
in the case of normal
cellular environment. Contradictory to this, tumor cells were
foundtobemoreresistanttoantioxidant(GSH)and
antioxidant enzyme levels (superoxide dismutase, peroxidase,
etc.), which were found to be depleted in UV-DMBA-induced
tumorigenic animals. Similarly, results were obtained when
astaxanthin was exposed to UV-A light; it was protected against
UV-A light induced oxidative stress in in vivo models when
compared with other carotenoids.
41−44
AX is a very efficient
antioxidant due to the unique structure of the terminal ring
moiety.
45,46
It is therefore feasible that AX has an affinity for the
superoxide free radical and thus may act as a satisfactory
antioxidant, ultimately preventing an increase in basal SOD
activity.
Increased retinol conversion (increased bioavailability) rate
by AXDE relative to AX (Figure 2) may offer enhanced
anticancer potential. Increased levels of AXDE and retinol in
the AXDE control groups of animals suggest that there is an
increased uptake of AXDE in vivo followed by AXME and
other carotenoids. Furthermore, within the bioavailable AX and
AXEs, maximum depletion of 1.4-fold of retinol was observed
in only AXDE followed by AXME, suggesting that AXDE may
Table 5. Effect of AX and AXEs on Antioxidant/Antioxidant Enzymes and TBARS Levels in Serum and Skin of UV-DMBA-
Induced Experimental Rats
a
SOD catalase GSH TBARS
group (U/mg protein) (nmol H2O2/mg protein) (μg GSH/mg protein) (μmol/MDA/mg protein)
Serum
healthy c 11.33 ±2.65 m 0.48 ±0.00 a 2.82 ±0.40 a 0.45 ±0.11 g
UV+DMBA 33.61 ±3.53 a 0.21 ±0.03 e 1.71 ±0.24 d 4.56 ±0.81 a
AX100*23.18 ±1.94 c 0.26 ±0.02 e 2.30 ±0.16 bc 3.11 ±0.13 c
AX200*19.04 ±2.01 f 0.31 ±0.03 d 2.28 ±0.44 c 3.22 ±0.12 c
AX200*c 13.36 ±3.46 j 0.35 ±0.07 c 2.34 ±0.17 bc 0.46 ±0.13 g
TC100*24.01 ±6.60 b 0.23 ±0.05 e 2.11 ±0.20 bc 3.60 ±0.10 b
TC200*19.89 ±1.13 e 0.34 ±0.03 c 2.17 ±0.41 bc 3.08 ±016 c
TC200*c 17.18 ±3.01 g 0.36 ±0.03 c 2.19 ±0.12 bc 0.47 ±0.06 g
AXME100*24.01 ±2.07 b 0.31 ±0.07 c 2.33 ±0.50 bc 2.34 ±0.23 d
AXME200*13.81 ±1.73 i 0.41 ±0.08 ab 2.41 ±0.36 a 2.15 ±0.21 d
AXME200*c 12.77 ±2.76 k 0.42 ±0.10 ab 2.94 ±1.84 a 0.46 ±0.05 g
AXDE100*20.73 ±0.58 d 0.29 ±0.01 cd 2.52 ±0.38 b 1.82 ±0.10 e
AXDE200*15.84 ±1.81 h 0.36 ±0.02 b 2.68 ±0.14 bc 1.59 ±0.23 f
AXDE200*c 11.58 ±2.20 l 0.45 ±0.10 a 2.73 ±0.14 a 0.35 ±0.10 g
Skin
healthy c 164.89 ±17.35 l 0.16 ±0.02 a 18.95 ±2.54 a 0.33 ±0.08 h
UV+DMBA 452.50 ±24.70 a 0.07 ±0.01 d 9.14 ±1.74 k 3.51 ±0.15 a
AX100*280.45 ±7.50 d 0.11 ±0.01 c 15.12 ±5.00 h 3.14 ±0.06 b
AX200*243.65 ±75.90 f 0.12 ±0.03 c 15.57 ±7.68 f 2.93 ±0.14 c
AX200*c 182.40 ±20.10 j 0.14 ±0.02 b 16.92 ±3.21 c 0.32 ±0.03 h
TC100*309.40 ±14.62 b 0.08 ±0.01 d 13.14 ±0.37 j 3.16 ±0.10 d
TC200*279.63 ±21.91 b 0.12 ±0.09 c 14.89 ±1.53 i 2.89 ±0.15 c
TC200*c 168.73 ±16.73 m 0.14 ±0.03 b 16.94 ±3.04 c 0.30 ±0.08 h
AXME100*292.02 ±19.50 c 0.11 ±0.04 c 14.50 ±0.60 i 2.39 ±0.03 d
AXME200*248.97 ±21.58 f 0.13 ±0.05 c 16.07 ±1.03 e 2.31 ±0.17 d
AXME200*c 208.08 ±30.27 i 0.15 ±0.03 a 16.97 ±4.81 c 0.68 ±0.14 g
AXDE100*275.09 ±26.21 e 0.12 ±0.06 c 15.38 ±3.77 g 2.16 ±0.06 e
AXDE200*223.12 ±10.36 h 0.13 ±0.02 c 16.66 ±4.31 d 1.90 ±0.10 f
AXDE200*c 173.04 ±13.11 k 0.16 ±0.02 a 17.03 ±0.68 b 0.31 ±0.10 h
a
Values are expressed as the mean ±SD. Values are not sharing a similar letter within the same column are significantly different (p< 0.05) as
determined by ANOVA. *,μg/kg bw. C,control; AX, astaxanthin; TC, total carotenoid; AXME, monoester of astaxanthin; AXDE, diester of
astaxanthin from H. pluvialis.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513848
be more bioavailable, yielding more retinol, which may be
utilized in animals during their exposure to UV-DMBA. Thus,
AXDE may confer better anticarcinogenic potency than other
compounds tested. AXEs may offer protection against skin
cancer at least partly by an antioxidative route, probably via
mediation of their metabolites rather than exhibiting anti-
oxidants by them, per se, and partly by tyrosinase inhibitory
potentials due to increased bioavailability of AXDE.
Our observed data are strongly encouraged by the
observations made previously by Camera et al.
12
and Savouré
et al.;
13
these two independent studies revealed that AX has
better anticancer potency than other carotenoids, canthaxanthin
and β-carotene. Furthermore, it was noted that UV-induced
skin cancer is via modifications of polyamine metabolism,
particularly by epidermal ornithine decarboxylase (ODC).
ODC induction was amplified by several-fold in the skin of
vitamin A-deficient animals relative to vitamin-normalized
animals and was not protected effectively by carotenoids.
However, AX had a stronger inhibitory effect than other
carotenoids on polyamine accumulation, suggesting an alternate
mechanism of protection against skin cancer besides retinol
activity by AX. Now with the observed results that AXEs are
more protective against cancer induction by UV-DMBA than
AX, it is important to address the role of AXEs on polyamine
accumulation in comparison with that of AX, which can further
strengthen the use of AXE-rich H. pluvialis against skin cancer.
Our observed data in the current paper indeed show the
most potent anticancer form of carotenoids probably better
than AX, although the latter by itself is more potent than other
reported carotenoids such as cantaxanthin and β-carotene.
Studies thus throw light on the possible application of AXEs
and AX-rich H. pluvialis if one compares the mechanism of
action of AX and AXEs with that of known anticancer drugs
and prognostic factors in various types of malignancies.
Furthermore, the neutrophil to lymphocyte ratio (NLR) has
Table 6. Effect of AX and AXEs on SGPT, SGOT, and SALP in the Serum and Skin of UV-DMBA-Induced Experimental Rats
a
SGPT SGOT SALP
group (U/mg protein) (U/mg protein) (U/mg protein)
Serum
healthy c 108.89 ±21.61 m 105.72 ±3.28 l 227.56 ±22.60 l
UV+DMBA 195.33 ±16.45 a 184.66 ±3.21 a 409.33 ±19.10 a
AX100*173.10 ±11.77 b 153.79 ±18.58 d 352.34 ±26.67 d
AX200*166.26 ±21.62 c 145.72 ±10.42 f 322.70 ±14.94 f
AX200*c 113.08 ±16.32 j 111.03 ±14.51 j 249.96 ±11.91 j
TC100*168.28 ±9.87 c 171.54 ±12.19 b 370.94 ±17.39 b
TC200*156.94 ±31.64 e 152.69 ±23.26 e 329.99 ±18.72 e
TC200*c 110.35 ±12.56 l 106.16 ±15.56 k 247.80 ±15.42 k
AXME100*161.98 ±13.55 d 159.03 ±19.17 c 359.33 ±12.90 c
AXME200*144.90 ±5.79 g 138.40 ±29.01 g 307.89 ±23.63 g
AXME200*c 111.91 ±12.55 jk 112.39 ±73.18 i 257.51 ±19.40 i
AXDE100*148.35 ±16.02 f 153.61 ±13.05 d 323.69 ±9.13 f
AXDE200*138.63 ±14.21 h 121.77 ±8.17 h 287.16 ±21.07 h
AXDE200*c 114.61 ±16.05 i 107.48 ±13.50 k 226.26 ±11.18 l
Skin
healthy c 72.68 ±15.06 fg 319.25 ±25.44 i 329.31 ±25.61 j
UV+DMBA 126.17 ±7.41 a 668.84 ±32.80 a 591.93 ±23.09 a
AX100*101.99 ±23.97 c 549.87 ±35.32 b 516.62 ±34.21 c
AX200*95.89 ±15.60 d 421.40 ±28.15 g 489.60 ±21.90 e
AX200*c 77.95 ±24.35 fg 295.92 ±15.61 j 390.18 ±15.23 h
TC100*115.05 ±8.13 b 547.59 ±18.82 b 528.50 ±25.12 b
TC200*95.23 ±6.14 b 495.16 ±21.93 c 498.76 ±31.03 d
TC200*c 75.05 ±27.48 g 316.12 ±36.88 i 365.06 ±34.49 j
AXME100*93.09 ±12.51 d 557.73 ±14.91 b 499.00 ±14.03 d
AXME200*82.85 ±12.97 e 484.05 ±11.51 c 442.59 ±12.10 f
AXME200*c 78.53 ±16.11 fg 311.27 ±16.29 i 378.39 ±25.78 i
AXDE100*100.49 ±29.97 c 536.06 ±18.92 b 484.47 ±14.27 e
AXDE200*79.75 ±3.09 f 447.20 ±3.85 f 418.47 ±18.25 g
AXDE200*c 71.58 ±14.42 h 331.53 ±17.30 h 378.64 ±20.95 i
a
Values are expressed as the mean ±SD. Values not sharing a similar letter within the same column are significantly different (p< 0.05) as
determined by ANOVA. *,μg/kg bw. C, control; AX, astaxanthin; TC, total carotenoid; AXME, monoester of astaxanthin; AXDE, diester of
astaxanthin from H. pluvialis.
Figure 5. Inhibition of skin tyrosinase activity in vitro by AX and
AXEs. AX, astaxanthin; TC, total carotenoid; AXME, monoester of
astaxanthin; AXDE, diester of astaxanthin from H. pluvialis. Each value
represents the mean ±SD (n= 3). Values not sharing a similar letter
between the groups are significantly different (p< 0.05) as determined
by one-way ANOVA. *,μg/kg bw.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513849
been documented as a simple index of systematic inflammatory
response in critically ill malignancy patients. Similarly, the
preoperative platelet to lymphocyte ratio (PLR) has been also
suggested as an independent significant prognostic indicator in
pancreatic cancer. In this perspective, AXDE was effective in
restoring normal levels of NLR and PLR ratios, suggesting the
role of AXDE as immunomodulator during inhibition of skin
cancer in the studied model.
With the observed results, therefore, Scheme 1 has been
proposed to explain the mechanism of protection of UV-
DMBA-induced cancer in animals by AX and AXEs of H.
pluvialis. Multistep action such as inhibition of accumulation of
ROS and inhibition of tyrosinase enzyme activity may result in
inhibition of UV-DMBA-induced skin cancer. Subsequently, the
mentioned properties may prevent uncontrolled proliferation of
melanocytes and prevention of accumulation of melanocytes
and melanin pigments in addition to the inhibition of
polyamine accumulation. Immunomodulatory action may
potentiate the anticancer property of H. pluvialis.
■AUTHOR INFORMATION
Corresponding Author
*Phone: +91-821-2514876. Fax: +91-821-2517233. E-mail:
shylaakshu@yahoo.com.
Funding
We acknowledge a research grant supported by the Department
of Science and Technology, government of India, New Delhi.
A.R.R. gratefully acknowledges the Indian Council of Medical
Research (ICMR), New Delhi, for the award of a Senior
Research Fellow.
Notes
The authors declare no competing financial interest.
■ABBREVIATIONS USED
AX, astaxanthin; AXEs, astaxanthin esters; AXME, astaxanthin
monoesters; AXDE, astaxanthin diesters; TC, total carotenoid;
UV, ultraviolet; DMBA, 7,12-dimethylbenz(a)anthracene;
TLC, thin layer chromatography; HPLC, high-performance
liquid chromatography; LC-MS, liquid chromatography−mass
spectrometry; APCI, atmospheric pressure chemical ionization;
ROS, reactive oxygen species; CAT, catalase; SOD, superoxide
dismutase; GSH, glutathione; MDA, malondialdehyde; TBA,
thiobarbituric acid; SGPT, serum glutamate pyruvate trans-
aminase; SGOT, serum glutamate oxaloacetate transaminase;
SALP, serum alkaline phosphatase; LYM, lymphocytes; PLT,
platelet count; HBCs, human buccal cells; NLR, neutrophil to
lymphocyte ratio; PLR, platelet to lymphocyte ratio; EDTA,
ethylenediaminetetraacetic acid; MDA, malondialdehyde;
TBARS, thiobarbituric acid reactive substances
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(A) Induces Skin Cancer (F) via Up-regulation of
Tyrosinase Enzyme in the Melanocyte
a
a
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Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf304609j |J. Agric. Food Chem. 2013, 61, 3842−38513851
