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Effective Inhibition of Skin Cancer, Tyrosinase and Antioxidative Properties by Astaxanthin and Astaxanthin Esters from Green Alga Haematococcus pluvialis.

  • Vignan`s Foundation for Science Technology and Research (Deemed to be University)

Abstract and Figures

Astaxanthin mono (AXME), di (AXDE) esters were characterized and examined for anticancer potency with total carotenoids (TC) and astaxanthin (AX) against UV-DMBA induced skin cancer model in rat. At 200 μg/kg b.w., AXDE and AXME reduced UV-DMBA induced tumor incidences up to 96% and 88% respectively when compared to those of AX (66%) and TC (85%). UV-DMBA has been known to generate high levels of free radicals and tyrosinase enzyme leading to characteristic symptom of skin pigmentation and tumor initiation. Intriguingly, ~ 7 and 10 folds of increased tyrosinase, decreased antioxidant levels were normalized by AXDE and AXME as opposed to ~ only 1.4 to 2.2 folds by AX and TC respectively. This result together with appearance of 72 and 58 ng/mL of retinol in the serum of respective AXEs (AXDE + AXME) and AX treated animals suggested that better anticancer potency of AXEs could be due to increased bioavailability.
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Eective 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 UV7,12-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 1.42.2-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
KEYWORDS: microalgae, AX, AXME, AXDE, UV-DMBA, skin cancer, retinol
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.
According to the World Cancer
Report, skin cancer constitutes 30% of all newly diagnosed
cancers in the world.
This rise in incidence has been attributed
to overexposure of skin to sun/UV light, due to reduction in
the ozone in the atmosphere.
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 signicantly to the
enhanced synthesis and accumulation of melanin in melano-
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.
Melanomas are
dicult 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,
from natural sources, to overcome chemoresistance and to
avoid side eects.
Indeed, much progress is being made in the
direction of pharmacological evaluation of various plant
products and dietary sources with the hope of achieving
eective chemoprevention.
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, 1520%
of diesters, and 45% of free forms, indicating the
predominance of esteried astaxanthin forms in H. pluvialis as
opposed to free forms in other plant sources.
activities 100 and 10 times greater than those of vitamin E and
β-carotene have been reported in AX.
Recently developed
downstream processing for the large-scale production of AX
and AXEs may potentiate their uses as anticancer alternatives.
Furthermore, intriguing studies by Camera et al.
and Savouré
et al.
implied that dierent carotenoids exhibit varied
potential to oer 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 eect toward photo-oxidative changes in
Received: October 30, 2012
Revised: March 10, 2013
Accepted: March 10, 2013
Published: March 11, 2013
© 2013 American Chemical Society 3842 |J. Agric. Food Chem. 2013, 61, 38423851
cell culture. Results of the present investigation add to the
previous observation that AXEs are more protective than AX in
UV7,12-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 rst 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 dened protocol for their optimization.
H. pluvialis.H. pluvialis was obtained from Sammlung von
Algenkulturen, Panzenphysiologisches Institute, Universitat Gottin-
gen, Gottingen, Germany, and was maintained on modied
autotrophic bold basal medium and agar slants.
Extraction, Isolation, and Characterization of AX and AXEs.
Total carotenoid (TC) from H. pluvialis biomass was extracted and
characterized as described previously.
Briey, 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 ow rate of 1.0 mL/min.
AX, AXEs, and
retinol were monitored at 476 and 325 nm with a UVvisible detector
(Shimadzu). Peak identication and λmax values of these components
were conrmed 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 quantied from their peak areas in relation to the respective
reference standards.
Characterization of AX and AXEs by Liquid Chromatog-
raphyMass 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/z4002000 scan range.
Eect 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-inducedgroup. 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
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.
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.
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
Tyrosinase enzyme activity was measured in the serum
and skin homogenates of all groups of animals using L-Dopa as
substrate with slight modications. L-Dopa (0.1 mL of a 1 mg/mL
solution) was mixed with 0.8 mL of 0.1 M phosphate buer (pH 6.0)
and incubated with 0.1 mL of serum/skin homogenates at 37 °C for
15 min. Dopachrome formation was measured uorometircally
(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.
Protein content was determined using the method described.
Superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)
levels and TBARS were measured as per the protocol described earlier
by our group.
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 xed in 10% buered formalin for 24
h. The processed tissues were embedded in paran blocks, and
sections made were stained with hematoxylin and eosin dye. The
sections were analyzed by observation under light microscope (Leitz,
Germany) at 10×magnication. 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
Determination of Bioavailability of AX and AXEs in Dierent
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.
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.
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 Duncans multiple-
range test at p< 0.05.
Characterization of AX and AXEs by HPLC and LC-MS
(APCI). The total carotenoid (TC) constituted about 23% 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 |J. Agric. Food Chem. 2013, 61, 384238513843
separated from total carotenoid extract of H. pluvialis with
acetone/hexane (3:7, v/v) mobile phase on silica-based thin
layer chromatography with dierent 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 identied by absorption spectra at 470476 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 reconrmed their
mobility with the same chromatographic system, and such pure
components isolated by preparative thin layer chromatography
were identied as AX and AXEs by LC-MS. LC-MS (APCI)
positive mode and the MS data used for the identication of
AX, AXME, and AXDE are summarized in Table 1. The mass
spectrum was obtained from an AXME (ME C16:0,MEC
ME C17:1,MEC
Because only mass dierences 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 identied 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 identied. The mass spectrum was obtained
from an AXDE (DE C16:0/C16:0,DEC
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.
Eect of TC, AX, and AXEs on UV-DMBA-Induced Skin
Carcinogenesis in in Vivo Model. Having identied
uniquely modied carotenoids such as esteried AX in a
natural source, H. pluvialis, and taking the fact that carotenoids
exhibit anticancer property dierentially, through dierent
mechanisms, the following were hypothesized in this study: (1)
the comparative anticancer ecacy of dierentially esteried
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) dierences
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 puried 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 eciency 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 inammatory patches. Skin
lesions with tumor nodules/tumor mass, angiogenesis, and
inammation 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 dierent levels of tumor reduction (Figure
1B,D,F,H,J). AXEs, however, showed the best inhibition with
8896% 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 dierential eects 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.;
the ecacy 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 ecacy of AX in oering
anticancer property. However, because AXEs appear to be
more potent, it would be interesting to compare their
pharmacokinetics to understand their practical feasibility.
Furthermore, it is also important to highlight here the
signicance of the slight modication in the methodology of
UV-DMBA-induced cancer in rats. Conventionally, signicant
levels of tumors were induced only after 2030 weeks
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 oers 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
no. [M] [M + 2H FA1]+1 MS2compound
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
ME, monoesters; DE, diesters.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 384238513844
2C,E,G,I). UV-DMBA-induced rats showed greater changes in
the epidermis and dermis including irregular distribution with
nger-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 oer better protection (Figure 2F,H)
than AX (Figure 2D). Tumor areas in control and treated
sample sections were localized using Image J software
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 Dierences in Tumor Incidences in
Healthy, UV-DMBA, and UV-DMBA+AX/AXE Samples
(%) mean tumor
burden (mm3)reduction in mean
tumor burden (%)
healthy c
UV-DMBA 100 a 748.84 ±89.13 a 0
AX200 c
TC200 c
AXME200 c
AXDE200 c
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
Each value represents the mean ±SD (n= 6). Values not sharing a
similar letter within the same column are signicantly dierent (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.
, 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 signicant epidermal thickness in the sections of UV-DMBA-induced
group when compared to healthy and sample control groups. *,μg/kg
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 384238513845
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
aected signicantly (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 dierentiation, 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 oer protection to
animals against UV-DMBA via its conversion to retinol. Similar
results were observed in the liver also.
Inhibitory Eect 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.
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. Eects
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 proles 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 signicantly 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 eects 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 signicant
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 signicance, 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
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
Each value represents the mean ±SD (n= 6). Values not sharing a similar letter within the same column are signicantly dierent (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 |J. Agric. Food Chem. 2013, 61, 384238513846
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 2.42.8-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 signicant dierence in the body weight gain prole, 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
signicant dierence either in the biochemical prole 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 eects on animals were found.
Melanoma is a relatively common and one of the most
malignant tumors in humans. The social impact of skin cancer
leading to melanoma is signicant because it has a very poor
more importantly, many melanoma cases occur in
young individuals, and there is little eective treatment available
once it becomes metastatic.
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.
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 diesteried
forms of astaxanthin of total carotenoids as opposed to either
free astaxanthin in other plant sources. It is of current interest
to nd out the bioactive potency of esteried astaxanthin in
comparison with astaxanthin alone. Dierences 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 rst time that AXE
exhibited a 3-fold higher potential in inhibiting skin cancer
incidences, and the ecacy 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 23-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 eect 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 signicantly dierent (p< 0.05) as determined by one-way ANOVA. *,μg/kg bw.
Table 4. Eect of AX and AXE on Tyrosinase Activity in
Serum and Skin Homogenates of UV-DMBA-Induced
Experimental Rats
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
Each value represents the mean ±SD (n= 6). Values not sharing a
similar letter within the same column are signicantly dierent (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 |J. Agric. Food Chem. 2013, 61, 384238513847
tumors were conrmed to be papillomas, whereas the extent of
hyperchromatia in the tumors of the rats that received AXEs
was observed to be approximately 23-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 benecial eect
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 eect is not directly proportional to the
antioxidant capacities because AXEs showed lowered antiox-
idant activity relative to AX and TC.
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 eect than the
lutein per se.
Also, observed results of the ecacy 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
in the case of normal
cellular environment. Contradictory to this, tumor cells were
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.
AX is a very ecient
antioxidant due to the unique structure of the terminal ring
It is therefore feasible that AX has an anity for the
superoxide free radical and thus may act as a satisfactory
antioxidant, ultimately preventing an increase in basal SOD
Increased retinol conversion (increased bioavailability) rate
by AXDE relative to AX (Figure 2) may oer 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. Eect of AX and AXEs on Antioxidant/Antioxidant Enzymes and TBARS Levels in Serum and Skin of UV-DMBA-
Induced Experimental Rats
SOD catalase GSH TBARS
group (U/mg protein) (nmol H2O2/mg protein) (μg GSH/mg protein) (μmol/MDA/mg protein)
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
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
Values are expressed as the mean ±SD. Values are not sharing a similar letter within the same column are signicantly dierent (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 |J. Agric. Food Chem. 2013, 61, 384238513848
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 oer 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.
and Savouré
et al.;
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 modications of polyamine metabolism,
particularly by epidermal ornithine decarboxylase (ODC).
ODC induction was amplied by several-fold in the skin of
vitamin A-decient animals relative to vitamin-normalized
animals and was not protected eectively by carotenoids.
However, AX had a stronger inhibitory eect 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. Eect of AX and AXEs on SGPT, SGOT, and SALP in the Serum and Skin of UV-DMBA-Induced Experimental Rats
group (U/mg protein) (U/mg protein) (U/mg protein)
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
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
Values are expressed as the mean ±SD. Values not sharing a similar letter within the same column are signicantly dierent (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 signicantly dierent (p< 0.05) as determined
by one-way ANOVA. *,μg/kg bw.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 384238513849
been documented as a simple index of systematic inammatory
response in critically ill malignancy patients. Similarly, the
preoperative platelet to lymphocyte ratio (PLR) has been also
suggested as an independent signicant prognostic indicator in
pancreatic cancer. In this perspective, AXDE was eective 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.
Corresponding Author
*Phone: +91-821-2514876. Fax: +91-821-2517233. E-mail:
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.
The authors declare no competing nancial interest.
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 chromatographymass
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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Scheme 1. Exposure of Experimental Animals to UV-DMBA
(A) Induces Skin Cancer (F) via Up-regulation of
Tyrosinase Enzyme in the Melanocyte
Increased exposure of normal melanocyte (B) to UV radiation and
DMBA (A) results in the aberrant melanocyte (C) due to increased
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Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 384238513851
... For example, Sarada et al. [78] assessed the extraction effectiveness of different technologies, in Arthrospira sp., the maximum extraction rate was achieved using freezethawing and homogenization, when combined with a hydrochloric acid pre-treatment; however, the pre-treatment led to a decrease in the purity of the compound. In the case of extraction of carotenoids, Dey and Rathod [79] optimised an ultrasound-assisted extraction of β-carotene from Arthrospira platensis, with optimal condition found using heptane at 30 • C and 167 W cm −2 . ...
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As a producer of pigments with known bioactive potential, cyanobacteria are a great source of active ingredients for cosmetics (i.e., carotenoids and phycobiliproteins). Multiple phases in the cyanobacteria-based bioprocess led to the obtention of these compounds. The marine Cyanobium sp. LEGE 06113 has been proposed as a promising source for pigments for cosmetic uses, and it has been optimized in the past few years in terms of production, extraction, and application of pigment extracts. This report aims at providing an overview of the cyanobacteria-based bioprocess, regarding optimization strategies, consolidating into a proposed bioprocess for this cyanobacterium. The optimization of Cyanobium sp. included strategies regarding its production (culture medium, light, temperature, pH and salinity) and extraction (successive solvent extraction and ohmic heating). After the optimization, the two pigment-rich extracts (carotenoids and phycobiliproteins) were assessed in terms of their cosmetic potential and compatibility as an ingredient. Finally, aiming a scale-up proposal, life cycle assessment (LCA) was used as tool for a sustainable process. Ultimately, the proposed process gives the possibility to obtain two stable cosmetic ingredients from the same biomass and applied as anti-agent agents, especially due to their high anti-hyaluronidase capacity. Moreover, there remain challenges and information regarding novel cosmetic ingredient regulations were also discussed.
... L-tyrosine and L-DOPA function as substrates and intermediates of melanin production (3). Alteration in melanin biosynthesis can lead to various skin diseases in humans (4). It has been known that plants with antioxidant properties might be utilized for the treatment of skin disorders. ...
Introduction: Tyrosinase is considered an important target of melanin biosynthesis inhibitors. Curcuma longa L. has been used in the Javanese traditional whitening cosmetics. This work aimed to explore the effect of C. longa extracts on mushroom tyrosinase activity and the cytotoxicity of the extract towards murine skin cancer B16F10 cells. Methods: C. longa rhizomes were cold-extracted using ethanol 70% and yielded 15.3% w/w of extract (ECL). The presence of curcuminoids in ECL was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). ECL was assessed for its inhibitory effects on mushroom tyrosinase activity using L-DOPA as substrate and kojic acid as the positive control drug. The cytotoxicity of ECL and curcumin was studied in B16F10 cells. Results: Triplet peaks of RP-HPLC chromatogram revealed that curcuminoids were available in ECL. The level of bisdemethoxycurcumin was 6.3306% (tR = 12.646 minutes), demethoxycurcumin was 3.1414% (tR = 13.675 minutes), and curcumin was 8.3754% (tR = 14.802 minutes). ECL had a weak inhibitory activity towards mushroom tyrosinase with IC50 = 564.8 µg/mL, while the IC50 = of kojic acid was 55.70 µg/mL. Both ECL and kojic acid had moderate toxicity to B16F10 cells (IC50 survival growth rates were 98.06 µg/mL and 65.54 µg/ mL, respectively). Curcumin was highly toxic to B16F10 cells (IC50 = 14.42 µg/mL). Conclusion: Taken together, ECL might be able to prevent melanogenesis via the inhibition of tyrosinase activity, and interestingly, it could inhibit the growth of murine skin cancer B16F10 cells. However, further studies are needed to verify its antimelanogenesis and anticancer properties.
... Due to its strong antioxidant activity, astaxanthin has attracted great attention from various industries including food, healthcare, pharmaceutical, cosmetic, and aquaculture industries (Ito et al. 2018;Lee et al. 2017;Saito et al. 2017). Moreover, astaxanthin helps to prevent cancer, protects from inflammation, and strengthens the immune system (McCall et al. 2018;Rao et al. 2013;Xie et al. 2020).In particular, natural astaxanthin comprises a large content of the 3S,3ʹS stereoisomer compared to other isomers, and its consumption is beneficial for humans as it provides 20 times greater free radical scavenging ability than artificial astaxanthin (Shah et al. 2016). Natural astaxanthin has been favored over chemically manufactured astaxanthin for widespread use in food supplements and clinical applications because synthetic astaxanthin has insufficient studies to validate its safety for human consumption (Brendler and Williamson 2019). ...
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Astaxanthin is receiving increasing interest as an antioxidant and high value-added secondary metabolite. Haematococcus pluvialis is the main source for astaxanthin production, and many studies are being conducted to increase the production of astaxanthin. In this study, we linked polyethylenimine (PEI) with chitosan to maintain astaxanthin-inducing ability while securing the recyclability of the inducer. Astaxanthin accumulation in H. pluvialis was induced to 86.4 pg cell⁻¹ with the PEI-chitosan fiber (PCF) treatment prepared by cross-linking of 10 μM PEI and low molecular weight (MW) chitosan via epichlorohydrin. PEI concentration affected the astaxanthin accumulation, whereas the MW of chitosan did not. In addition, the PCF treatment in H. pluvialis increased the reactive oxygen species (ROS) content in cells, thereby upregulating the transcription of enzymes involved in astaxanthin biosynthesis. PCF can be reused multiple times with the maintenance of over 90% of the astaxanthin production efficiency. This study offers a reusable PCF stimulation strategy for enhancing natural astaxanthin content, and PCF treatment will easily increase the production scale or reduce production costs by using recyclability that is not available in current methods. Key points • Polyethylenimine-chitosan fiber (PCF) was applied to Haematococcus pluvialis • PCF promotes astaxanthin accumulation by enhancing oxidative stress in H. pluvialis • PCF can be reused multiple times with maintaining over 90% production efficiency
... 70 Astaxanthin exhibits strong antioxidant effects and antilipid per-oxidation activity because of its unique molecular structure bearing two hydroxy substituents at positions 3 and 3′ (the 3S, 3′S diastereomer), which enable it to present both inside and outside of cell membrane. 71 In addition to antioxidant and anti-inflammatory properties, 72,73 astaxanthin has proven its neuroprotective effects on pathological features of AD, evidenced by in vitro and in vivo studies., 74,75 However, no substantial evidence was obtained from human studies regarding the role of astaxanthin in AD pathogenesis. The dysfunction of the blood-brain barrier (BBB) found in AD pathological changes could range from minor disruption of tight junctions with increased BBB leakiness to chronic loss in integrity with an altered transport of molecules across the brain hypo-perfusion and inflammatory activation. ...
Alzheimer's disease (AD) is a neurodegenerative disorder of an ever-increasing aging population with various pathological features such as β-amyloid (Aβ) aggregation, oxidative stress, an impaired cholinergic system, and neuroinflammation. Several therapeutic drugs have been introduced to slow the progression of AD by targeting the above-mentioned pathways. In addition, emerging evidence suggests that naturally occurring compounds have the potential to serve as adjuvant therapies to alleviate AD symptoms. Carotenoids, a group of natural pigments with antioxidative and anti-inflammatory properties, are proposed to be implicated in neuroprotection. To obtain a comprehensive picture of the effect of carotenoids on AD prevention and development, we critically reviewed and discussed recent evidence from in silico, in vitro, in vivo, and human studies in databases including PubMed, Web of Science, Google Scholar, and Cochrane (CENTRAL). After analyzing the existing evidence, we found that high-quality randomized controlled trials (RCTs) are lacking to explore the neuroprotective role of carotenoids in AD pathogenesis and symptoms, especially carotenoids with solid preclinical evidence such as astaxanthin, fucoxanthin, macular carotenoids, and crocin, in order to develop effective preventive dietary supplements for AD patients to ameliorate the symptoms. This review points out directions for future studies to advance the knowledge in this field.
... It is capable of neutralizing the amount of singlet oxygen. It also modulates the gene function and immune function (Gentili et al., 2013;Rao et al., 2013). So, it could be helpful for the reduction of several oxidative markers in the human body. ...
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Dark chocolate gets popularity for several decades due to its enormous health benefits. It contains several health-promoting factors (bioactive components - polyphenols, flavonoids, procyanidins,theobromines, etc, and vitamins and minerals) that positively modulate the immune system of human beings. It confers safeguards against cardiovascular diseases, certain types of cancers, and other brain-related disorders like Alzheimer's disease, Parkinson's disease, etc. Dark chocolate is considered a functional food due to its anti-diabetic, anti-inflammatory, and anti-microbial properties. It also has a well-established role in weight management and the alteration of a lipid profile to a healthy direction. But during the processing of dark chocolate, several nutrients are lost (polyphenol, flavonoids, flavan 3 ol, ascorbic acid, and thiamine). So, fortification would be an effective method of enhancing the overall nutrient content and also making the dark chocolate self-sufficient. Thus, the focus of this review study is to gather all the experimental studies done on dark chocolate fortification. Several ingredients were used for the fortification, such as fruits (mulberry, chokeberries, and elderberries), spices (cinnamon), phytosterols, peanut oil, probiotics (mainly Lactobacillus, bacillus spices), prebiotics (inulin, xanthan gum, and maltodextrin), flavonoids, flavan-3-ols, etc. Those fortifications were done to raise the total antioxidant content as well as essential fatty acid content simultaneously reducing total calorie content. Sometimes, the fortification was done to improve physical properties like viscosity, rheological propertiesand also improve overall consumer acceptance by modifying its bitter taste.
... They are often more orally bioavailable than astaxanthin in lipid-based formulations and have better thermal stability [106]. Some astaxanthin ester derivatives (e.g., astaxanthin mono and diesters) obtained from green algae (e.g., Haematococcus pluvialis) even show improved antitumor effect against UV-7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer model in rat [107]. ...
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Melanoma cells are highly invasive and metastatic tumor cells and commonly express molecular alterations that contribute to multidrug resistance (e.g., BRAFV600E mutation). Conventional treatment is not effective in a long term, requiring an exhaustive search for new alternatives. Recently, carotenoids from microalgae have been investigated as adjuvant in antimelanoma therapy due to their safety and acceptable clinical tolerability. Many of them are currently used as food supplements. In this review, we have compiled several studies that show microalgal carotenoids inhibit cell proliferation, cell migration and invasion, as well as induced cell cycle arrest and apoptosis in various melanoma cell lines. MAPK and NF-ĸB pathway, MMP and apoptotic factors are frequently affected after exposure to microalgal carotenoids. Fucoxanthin, astaxanthin and zeaxanthin are the main carotenoids investigated, in both in vitro and in vivo experimental models. Preclinical data indicate these compounds exhibit direct antimelanoma effect but are also capable of restoring melanoma cells sensitivity to conventional chemotherapy (e.g., vemurafenib and dacarbazine).
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Astaxanthin is one of the most effective and potent anti-oxidants astaxanthin is also a natural source for pigmentation in several aquatic organisms. Its utility to impart bright red coloration in farmed aquaculture animals is well recognized. In addition, astaxanthin has potential benefits to aquaculture species such as increasing their growth, survivability, improving flesh quality, and boosting reproductive performance and egg quality. Moreover, among of the many immunopotentiators, astaxanthin is more effective and an environmentally-friendly natural source mostly utilized in different fish diets for improving their immunological, hematological, and antioxidant properties. The demand for natural astaxanthin is also increasing in the poultry industries because of its potential in enhancing growth, immunity and pigmentation as well as the quality of both meat and egg. The green alga, Haematococcus pluvialis has received much attention for the production of astaxanthin on an industrial scale. Furthermore, Monoraphidium is another green alga that has potential for astaxanthin production. Furthermore, Chlorella zofingiensis, Chlorococcum spp., Scenedesmus spp., Chlamydomonas nivalis, Nannochloropsis spp., Chlamydocapsa spp., Chlorella vulgaris, Eremosphaera viridis, Neochloris wimmeri and Coelastrella striolata are also possible sources of astaxanthin. This review summarizes the potential microalgal sources of astaxanthin as well as downstream processing and the utilization of astaxanthin in the aquaculture and poultry industries.
Extracellular tissue fluids are interesting biomatrices that have recently attracted scientists' interest. Many significant biomarkers for localized external organ diseases have been isolated from this biofluid. In the diagnostic and disease monitoring context, measuring biochemical entities from the fluids surrounding the diseased tissues might give more important clinical value than measuring them at a systemic level. Despite all these facts, pushing tissue fluid‐based diagnosis and monitoring forwards to clinical settings faces one major problem: its accessibility. Most extracellular tissue fluid, such as ISF, is abundant but hard to collect, and the currently available technologies are invasive and expensive. This is where novel microneedle technology could help tackle this significant obstacle. The ability of microneedle technology to minimally invasively access tissue fluid‐containing biomarkers will enable ISF and other tissue fluid utilization in the clinical diagnosis and monitoring of localized diseases. This review attempts to present the current pursuit of the application of microneedle systems as a diagnostic and monitoring platform, along with the recent progress of biomarker detection in diagnosing and monitoring localized external organ diseases. Then, the potential use of various microneedles in future clinical diagnostics and monitoring of localized diseases is discussed by presenting the currently studied cases. This article is protected by copyright. All rights reserved
Background Astaxanthin is an important commercially valuable secondary metabolite produced during fermentation of the yeast Phaffia rhodozyma, which can be used in aquatic feedstock. Results In this study, we used sweet potato juice (SPJ), the by-product of sweet potato starch, to culture P. rhodozyma of high-density and confirmed that hydrochloric acid-deproteinized sweet potato juice is suitable for the culture of P. rhodozyma, and using this as a substrate, supplemented with 0.05% yeast extract, we performed batch fermentation in a 5-L fermenter. Compared with shaking flask fermentation, we obtained an 18.86% increase in yeast biomass and a 32.5% increase in astaxanthin yield using the batch process. After culturing P. rhodozyma in a 5-L fermenter for 120 h, we achieved biomass and an astaxanthin yield of 45.2 g/L and 19.465 mg/L, respectively. Conclusions In this study, we found that deproteinized SPJ was the most suitable for P. rhodozyma through experiments on different cultivars and different processing stages of SPJ. On the basis of deproteinized SPJ, supplemented with yeast extract, the biomass and astaxanthin yield reached a high level. The optimized system can substantially reduce the costs of raw material for astaxanthin yield by P. rhodozyma and enhance the comprehensive utilization of sweet potato resources.
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Background: Astaxanthin (ASTA) is a fat-soluble xanthophyll with powerful antioxidant functions. It is extracted from e.g. salmon, an important food source for certain human populations known to have a reduced risk of tumor development. It is possible that ASTA plays a role in cancer chemoprevention in such populations. The purpose of this study was to investigate the effects of dietary ASTA on chemically induced mammary tumorigenesis using N-methyl-N-nitroso-urea (MNU) in immature Wistar rats. Methods: Thirty-six 37 days old juvenile female Wistar rats were at random allocated to 4 groups of which Groups 1 and 2 received a single dose of 55 mg MNU/kg body weight. The effects of ASTA was evaluated by giving rats of Groups 2 and 4 a dose of 50 mg ASTA/kg/day for the entire duration of the study. Group 3 rats received feed added alimentary oil.Necropsy and histopathological examinations were carried out on each rat 14 months after the administration of MNU. Haematological values and antioxidative status were determined. Oxidative stress was evaluated by monitoring superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in hepatic tissue. Lipid peroxidation and carbonylation of proteins was determined in protein extracts from the liver. Results: Tumor development occurred only in rats of Groups 1 and 2, i.e. MNU exposed animals. Frequency of tumor development in general and average number of tumors per animal were insignificant between these two groups. Mammary gland tumors developed in equal frequencies in Group 1 and 2 rats, respectively. Although only rather few tumors were found in the mammary glands, a substantial number of other tumors were found in Group 1 and 2 rats, but at equal rates.Biochemical analyses showed significant higher levels of GPx, malondialdehyde and dinitrophenylhydrazine in Group 1 rats that for rats in all other groups thus indicating protective effects of ASTA on MNU induced hepatic oxidative stress. Conclusions: Supplementation with ASTA did not reduce tumorigenesis induced by MNU in Wistar rats. However, supplementation with ASTA seemed to have anti-inflammatory effects.
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The influence of stress was studied on astaxanthin production in Haematococcus pluvialis grown under different culture conditions. High concentrations of NaCl (>1.0% w/v) were lethal and the age of the culture was crucial for stress induced astaxanthin production. Four to-eight-day-old cultures were sensitive to NaCl addition while older cultures (12–16 days) were resistant. Older cells accumulated 8.3–10.69 mg/l astaxanthin compared to 0.95–8.1 mg/l in 4–8-day-old cultures, respectively. Cultures grown with calcium nitrate as nitrogen source showed a significant increase in astaxanthin content and production under stress compared to other nitrogen sources. Similarly, cultures grown at pH 7.0 showed more astaxanthin production than those grown at pHs of 6.0, 8.0 and 9.0.
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The effects of the carotenoids β-carotene and astaxanthin on the peroxidation of liposomes induced by ADP and Fe2+ were examined. Both compounds inhibited production of lipid peroxides, astaxanthin being about 2-fold more effective than β-carotene. The difference in the modes of destruction of the conjugated polyene chain between β-carotene and astaxanthin suggested that the conjugated polyene moiety and terminal ring moieties of the more potent astaxanthin trapped radicals in the membrane and both at the membrane surface and in the membrane, respectively, whereas only the conjugated polyene chain of β-carotene was responsible for radical trapping near the membrane surface and in the interior of the membrane. The efficient antioxidant activity of astaxanthin is suggested to be due to the unique structure of the terminal ring moiety.
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Plant extracts are the most attractive sources of newer drugs and have been shown to produce promising results for the treatment of gastric ulcers. Karanjin, a furano-flavonoid has been evaluated for anti-ulcerogenic property by employing adult male albino rats. Karanjin (>95% pure) was administered to these rats in two different concentrations, that is, 10 and 20 mg kg(-1) b.w. Ulcers were induced in the experimental animals by swim and ethanol stress. Serum, stomach and liver-tissue homogenates were assessed for biochemical parameters. Karanjin inhibited 50 and 74% of ulcers induced by swim stress at 10 and 20 mg kg(-1) b.w., respectively. Gastric mucin was protected up to 85% in case of swim stress, whereas only 47% mucin recovery was seen in ethanol stress induced ulcers. H(+), K(+)-ATPase activity, which was increased 2-fold in ulcer conditions, was normalized by Karanjin in both swim/ethanol stress-induced ulcer models. Karanjin could inhibit oxidative stress as evidenced by the normalization of lipid peroxidation and antioxidant enzyme (i.e., catalase, peroxidase and superoxide dismutase) levels. Karanjin at concentrations of 20 mg kg(-1) b.w., when administered orally for 14 days, did not indicate any lethal effects. There were no significant differences in total protein, serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase and alkaline phosphatase between normal and Karanjin-treated rats indicating no adverse effect on major organs. During treatment schedule, animals remained as healthy as control animals with normal food and water intake and body weight gain.
Cancer chemoprevention is the use of specific natural or synthetic substances with the objective of reversing, suppressing, or preventing carcinogenic progression to invasive cancer Currently, numerous chemopreventive agents are in various stages of development and testing. Part I of this two-part series provides an overview of issues unique to chemoprevention trials, including the use of surrogate biomarkers as endpoints. This is followed by a discussion of the retinoids, such as all-trans-retinoic acid (ATRA [Vesanoid]), 9-cis-retinoic acid (9cRA), and isotretinoin (Accutane), and the carotenoids (eg, beta-carotene and lycopene) and other "classic" antioxidants (eg, vitamins E and C and selenium), Research oil these agents will be delineated by disease site when applicable. Part 2, which will appear in next month's issue, will focus on hormonally mediated chemopreventive agents, such as tamoxifen (Nolvadex), finasteride (Proscar), oral contraceptives, and dehydroepiandrosterone (DHEA). Part 2 also will cover nonantioxidant natural agents, such as calcium, the polyphenols, the isothiocyanates, and genistein; nonsteroidal anti-inflammatory drugs (NSAIDS), such as celecoxib, sulindac sulfone, and aspirin; difluromethylornithine (DFMO [Eflornithine]); oltipraz; and N-acetylcysteine.
Cuminaldehyde (p-isopropylbenzaldehyde) was identified as a potent mushroom tyrosinase inhibitor from cumin, a common food spice. This benzaldehyde derivative was found to inhibit the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase with an ID50 of 7.7 μg/mL (0.05 mM). Its oxidized analogue, cumic acid (p-isopropylbenzoic acid), was also characterized to inhibit this oxidation with an ID50 of 43 μg/mL (0.26 mM). These two inhibitors affect mushroom tyrosinase activity in different ways. Keywords: Cumin; cuminaldehyde; cumic acid; tyrosinase inhibitory activity; noncompetitive inhibition; Schiff base formation
Antioxidants are now being incorporated into sunscreens as additional topical measure for delaying the aging process and reducing photo-damage to skin induced by excessive UVA exposure. UVA radiation reaching the skin leads to the generation of ROS (reactive oxygen species) implicated in DNA damage and activation of matrix metalloproteinase-1 (MMP-1) responsible for collagen damage and photo-aging. Nitroxides are a class of compounds endowed with versatile antioxidant activity and recently, nitroxide-based UV filters in which a nitroxide moiety has been attached to the most popular UV filter present in sunscreens have been developed. This study explores the potential photo-protective effects of these compounds on ROS production and induction of MMP-1 in cultured human dermal fibroblasts exposed to UVA. For comparison, vitamin E was also tested. The effects were assessed by measuring intracellular ROS production using a ROS-index probe and MMP-1 mRNA expression levels using quantitative real-time PCR (qPCR). Exposure of fibroblasts to 18J/cm(2) UVA lead to a two-fold increase in ROS production which was reduced to non-irradiated control levels in the presence of 50μM nitroxide compounds and vitamin E. Under the same conditions, a ten-fold increase in MMP-1 mRNA expression levels was observed 24h post-UVA treatment which was significantly reduced by all nitroxide compounds but not vitamin E. The results of this study support the potential use of nitroxide compounds, including novel nitroxide-based UV filters, as a useful and alternative strategy for improving the efficacy of topical formulations against photo-aging and possibly photo-carcinogenesis.