Food Chemistry 134 (2012) 308–314

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evaluation of antioxidant activity of carotenoid extract from shrimp processing

byproducts by in vitro assays and in membrane model system
R. Sowmya, N.M. Sachindra ⇑
Department of Meat, Fish and Poultry Technology, Central Food Technological Research Institute (Council of Scientific and Industrial Research), Mysore 570020, India

a r t i c l e i n f o a b s t r a c t

Article history: Carotenoid extracts from shrimp processing discards were evaluated for antioxidant activity. Crude
Received 7 December 2011 extract and fractions rich in astaxanthin showed strong antioxidant activity as indicated by radical
Received in revised form 8 February 2012 scavenging, reducing activity and metal chelating activity, comparable to that of the known
Accepted 23 February 2012
antioxidants a-tocopherol and TBHQ. Singlet oxygen quenching activity of crude extract and its fractions
Available online 3 March 2012
was higher than that of a-tocopherol. Nitric oxide scavenging activity was also higher than a-tocopherol.
The ability of the astaxanthin-rich fraction to inhibit the thermal oxidation of phospholipid liposomes,
Keywords:
with a protection factor of 22.6 ± 1.7 units, was better than that of a-tocopherol (8.5 ± 1.5 units). The
Carotenoid
Shrimp
higher antioxidant activity of the astaxanthin-rich fraction was also indicated by a higher protection fac-
Radical scavenging tor (14.1 ± 3.2 units) compared to a-tocopherol (6.2 ± 0.1 units) against singlet oxygen mediated oxida-
Singlet oxygen quenching tion of liposomes. The results indicate the potential of shrimp carotenoid extract as a natural
Liposome antioxidant for possible use in food and biomedical applications.
Antioxidant Ó 2012 Elsevier Ltd. All rights reserved.
Oxidation

1. Introduction
One of the important characteristics of carotenoids is their
ability to act as antioxidants, thus protecting cells and tissues from
damaging effects of free radicals and singlet oxygen. The free radi-
cals and singlet oxygen produced in the body by normal aerobic
metabolism can react with various components of living cells and
cause structural changes, leading to diseases, such as ageing (Ames
& Shigenga, 1992), atherogenesis (Esterbauer, Gebicki, Puhl, &
Jurgens, 1992), ischaemia (Takayama, Egashiru, Kudo, & Yamanaka,
1992), infant retinopathy (Phelps, 1987) and carcinogenesis
(Breimer, 1990). Carotenoids have been found to be important in
protecting against diseases and age-related phenomena caused by
oxidants (Halliwell, 1996).
Singlet oxygen is one of the important reactive oxygen species
implicated in diseases. Carotenoids have the ability to quench sin-
glet oxygen and to scavenge free radicals (Hirayama, Nakamura,
Hamada, & Kobayashi, 1994). Several natural antioxidants are
known to quench singlet oxygen, both by physical and chemical
mechanisms (Mukai, Itoh, Daifuku, Morimoto, & Inoue, 1993).
Carotenoids are known to be stronger singlet oxygen quenchers
than a-tocopherol (Sachindra et al., 2007). The singlet oxygen
quenching ability of several carotenoids has been studied. Lyco-
pene was found to be an effective singlet oxygen quenching
⇑ Corresponding author. Tel.: +91 821 2514840; fax: +91 821 2517233.
E-mail address: sachiprathi@yahoo.com (N.M. Sachindra).
carotenoid (Tinkler, Bohn, Schalch, & Truscott, 1994). Singlet oxy-
gen quenching activity of astaxanthin was found to be double than
that of b-carotene and 80 times more than the antioxidant a-
tocopherol (Di Mascio, Murhy, & Sies, 1991). Astaxanthin is dem-
onstrated to be a better agent for destroying free radicals than
other carotenoids (Nielsen, Mortensen, Jorgensen, & Skibsted,
1996).
Carotenoids occur widely in nature. Crustacean exoskeleton is
an important natural source of carotenoids, particularly astaxan-
thin. Shrimps are generally harvested using trawl nets and large
quantities of shrimp are processed in seafood export processing
units, generating large quantities of waste in the form of head
and body carapace. Utilisation of such large quantities of shrimp
processing discards for recovery of bioactive molecule such as
carotenoids would not only reduce the disposal problems associ-
ated with these wastes, but also enhance the economy of shrimp
processing. Several studies have been carried out to recover the
pigment from crustacean processing discards. Methods such as
extraction of carotenoids using organic solvents and edible oils
have been attempted (Sachindra, Bhaskar, & Mahendrakar, 2006;
Sachindra & Mahendrakar, 2006). Few reports are available on
the antioxidant activity of carotenoid-containing shrimp extracts.
Studies have been carried out on the antioxidant activity of
carotenoid-containing protein isolate from crustacean wastes.
Lyophilised fermentation liquor from Indian shrimp waste was
found to be rich in carotenoids and exhibited strong antioxidant
activity (Sachindra & Bhaskar, 2008). Carotenoprotein isolated

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.02.147
 R. Sowmya, N.M. Sachindra / Food Chemistry 134 (2012) 308–314
from shrimp waste by adopting pH shift technique was found to
have strong DPPH-scavenging activity (Meenata, Sowmya,
Rathinaraj, & Sachindra, 2011). Sowmya, Rathinaraj, and Sachindra
(2011) optimised the conditions for isolation of antioxidant-rich
carotenoprotein from shrimp heads using an autolytic process.
Studies on antioxidant activity of carotenoids are restricted to
the pure form of carotenoids. Shrimp exoskeleton contains asta-
xanthin and its esters as the major pigments (Sachindra, Bhaskar,
& Mahendrakar, 2005a, 2005b; Sachindra et al., 2006). Presence
of other antioxidants also influences the antioxidant potential of
carotenoids (Haila, Lievonen, & Heinonen, 1998) and other antiox-
idants such as tocopherols protect the carotenoids against radical
autooxidation (Heinonen, Haila, Lampi, & Piironen, 1997). It is also
important to study the behaviour of antioxidants in membrane
systems. Liposomes have been used to evaluate the behaviour of
carotenoids in membrane model systems. Comparison of the anti-
oxidant activity of polar carotenoids including astaxanthin and
astaxanthin-b-glucoside from marine bacteria on PC liposomes
indicated that they are highly active antioxidants (Matsushita,
Suzuki, Nara, Yokoyama, & Miyashita, 2000).
With this background, the antioxidant activity of a crude carot-
enoid extract from shrimp waste and its different fractions was
studied using various in vitro assays and also its ability to prevent
the thermally and singlet oxygen-mediated oxidation of egg-yolk
PL liposomes.
2. Materials and methods
2.1. Materials
Head and carapace of Penaeus indicus was collected from a local
market, and transported to the laboratory under chilled conditions.
The material was homogenised in a table top vertical cutter
(Robot-Coupe R10, Robot-Coupe, France) before use. 2,20 -
Diphenylpicrylhydrazyl (DPPH), 2,20 -azinobis-3-ethylbenzothiazo-
line-6-sulphonate (ABTS), luminal and standard astaxanthin were
procured from Sigma. All the other chemicals were of from M/s
SRL, India.
2.2. Preparation of shrimp waste extract
Carotenoids from shrimp waste (100 g) were extracted by
homogenising the sample with 100 ml of acetone. The extract
was filtered and the residue was repeatedly extracted with fresh
solvent and the filtrate collected until it was colourless. The solvent
extracts were pooled together and were phase separated with an
equal quantity of hexane. The hexane extract was repeatedly
washed with an equal quantity of 0.1% saline to remove traces of
acetone if any, and dried with 25 g of sodium sulphate, filtered
and then evaporated under vacuum at 40 °C using a rotary flash
evaporator and flushed with nitrogen to obtain the crude extract
(CE).
2.2.1. Fractionation of carotenoids
The crude extract containing carotenoids was fractionated by
silica gel column chromatography by the method of Lin, Chen,
and Chen (2005) with slight modifications. The concentrated crude
extract was dissolved in hexane and was applied to a column,
packed with Silica Gel G (60–120 mesh) and eluted initially with
100% hexane to obtain hexane fraction (F1), hexane:acetone
(92:8) to obtain astaxanthin esters (F2) and finally with hex-
ane:acetone (80:20) to obtain astaxanthin fraction (F3). All the
fractions were evaporated in a rotary evaporator and flushed with
nitrogen and stored at 20 °C till use.
309
2.2.2. Determination of carotenoid content
A known quantity of the carotenoid concentrate (CE, F1, F2 and
F3) was dissolved in hexane and made up to 100 ml and the absor-
bance of the appropriately diluted extract was measured at
468 nm. The carotenoid content was calculated as astaxanthin
(Simpson & Haard, 1985) using the equation,
Carotenoid contentðlg astaxanthin=g sampleÞ
A468nm  V extract  Dilution factor
¼
0:2  W sample
where, A is absorbance, V is volume of extract and 0.2 is the A468 of
1 lg/ml of standard astaxanthin and W is weight of extract in
grams.
2.2.3. High performance liquid chromatography of carotenoids
HPLC separation of crude carotenoid extract and fractions was
performed using TSK gel ODS 80 TS column (4.6 mm  25 cm).
The separation was carried out at flow rate of 1.25 ml/min with a
7:3 (A:B) ratio of the following mobile phase.
Solvent A: methanol:water (95:5).
Solvent B: tetrahydrofuran:methanol (3:7).
The detection was carried out at 450 nm. Carotenoid standards
were run for determining the retention time (Rt) and to compare
the Rt of individual fractions and crude extract with that of the
standards. The astaxanthin content in samples was calculated form
the standard curve of astaxanthin.
2.3. In vitro antioxidant assay
2.3.1. DPPH radical-scavenging activity
DPPH radical-scavenging activity of the samples (CE, F1, F2 and
F3) was measured by the method described by Duan, Zhang, Li, and
Wang (2006). An aliquot of sample was made up to 2 ml with
methanol and mixed with 2 ml of 0.16 mM DPPH in methanol
and incubated at 37 °C for 30 min in the dark. Methanol alone in
the reaction mixture served as control. Sample blank was prepared
by replacing the DPPH with methanol. The absorbance of the sam-
ple after incubation was measured at 517 nm and the scavenging
activity was calculated as follows:
Scavenging% ¼ ð1  ðAsample  Asample blank Þ=Acontrol ÞÞ  100
Scavenging activity was represented as mg TBHQ equivalent/
mg sample, using TBHQ standard curve.
2.3.2. ABTS radical scavenging assay
ABTS radical-scavenging activity was determined as explained
by Sachindra et al. (2007). ABTS radical solution was prepared by
mixing 5 ml of ready-to-use ABTS solution with 100 ml of acetate
buffer (0.05 M, pH 4.5) and 5 units of peroxidase and incubating
at 37 °C for 15 h. The decolorisation of the ABTS radical solution
was initiated by mixing 250 ll of ABTS solution with 25 ll of sam-
ple and incubating at 37 °C for 1 h in a 96-well microplate. To the
sample blank, 250 ll of acetate buffer (pH 4.5, 0.05 M) were added
instead of ABTS. ABTS solution without sample served as control.
The absorbance was measured at 405 nm initially and at the end
of the incubation period. Scavenging activity was calculated as
follows:
Scavengingð%Þ ¼ ½1  ðAsample  Asample blank Þ=Acontrol   100
ABTS radical scavenging activity was represented as mg TBHQ
equivalent/mg sample, using a TBHQ standard curve.
310 R. Sowmya, N.M. Sachindra / Food Chemistry 134 (2012) 308–314
2.3.3. Nitric oxide scavenging
Nitric oxide scavenging activity was measured by the methods
of Tsai, Tsai, Yu, and Ho (2007). An aliquot of the sample was incu-
bated with 10 mM sodium nitroprusside at 25 °C under light for
half an hour and nitric oxide scavenged was measured in terms
of nitrite present by adding Griess reagent and measuring
absorbance at 540 nm. Scavenging percentage was calculated
as mentioned above. NO scavenging was represented as mg
a-tocopherol equivalent/mg sample.
2.3.4. Hypochlorite scavenging
Hypochlorite scavenging activity was determined by a modified
method of Learn, Brestel, and Seetharama (1987). To an aliquot of
the sample, 100 ll of 0.1 mM luminol, and 20 ll of 4 mM NaOCl
were added. Luminescence was measured immediately following
the addition of 100 ll of 4 mM H2O2. Total luminescence count
(L) was used to calculate the scavenging as follows,
HClO  scavengingð%Þ ¼ ½ðLcontrol  Ltest Þ=Lcontrol   100
Scavenging was represented as mg gallic acid equivalent/mg
sample.
2.3.5. Singlet oxygen quenching
An aliquot of the sample (3–5 lg) was made up to 30 ll with
DMSO. To this 30 ll of 80 mM KBr, 30 ll of 0.4% H2O2 and 30 ll
of 0.8 mM luminol were added. Luminescence was measured
following the addition of 30 ll of peroxidase (10 lg/ml) (Wada et
al., 2007).
Quenchingð%Þ ¼ ðCLcontrol  CLsample Þ=CLcontrol Þ  100
Singlet oxygen quenching was represented as mg a-tocopherol
equivalent/mg sample.
2.3.6. Reducing activity
Reducing activity of extracts was determined as explained by
Kuda, Hishi, and Maekawa (2006). An aliquot of the sample was
made up to 200 ll with phosphate buffer of pH 6.6. To this sample
0.2 ml of 1% potassium ferricyanide were added and incubated at
50 °C for 20 min. After incubation, 0.2 ml of 10% TCA, 0.6 ml of dis-
tilled water and 0.1% of ferrous chloride were added and the absor-
bance was measured at 650 nm. Reducing activity was represented
as ascorbic acid equivalent (mg/mg sample) by referring to ascor-
bic acid standard curve.
2.3.7. Metal chelating activity
To an aliquot of the sample, 0.5 mM ferrous chloride and
2.5 mM ferrozine were added and incubated for 20 min (Dinis,
Madeira, & Almeida, 1994). Absorbance was measured as
562 nm. Percentage decrease in absorbance was measured by com-
paring with the control. The activity was represented as EDTA
equivalents (lg/mg sample) by referring to EDTA standard curve.
2.4. Inhibition of liposome oxidation
2.4.1. Preparation of liposome
Liposome suspension with carotenoids was prepared by mixing
egg yolk phospholipids (PL) purified by the method of Palacios and
Wang (2005). Stock solution of PL in chloroform (final concentra-
tion of 2.5 mg/ml) was mixed with 2.5 mg of sample in methanol,
evaporated to dryness by flushing with nitrogen. Distilled water
(10 ml) was added to the dried film and sonicated for 5 min at
50 Hz, with 1:1 pulse frequency using a sonicator.
2.4.2. Inhibition of thermal oxidation
Liposome suspensions containing different extracts (CE, F1, F2
and F3) along with control were incubated at 50 °C under light.
The samples were drawn at intervals of 0, 2, 4, 6, 8 and 24 h for
determination of carotenoid and conjugated diene content. Carote-
noids from the liposome were extracted by mixing 1 ml of suspen-
sion with 3 ml of solvent mixture of ethanol, diethyl ether and
hexane in the ratio of 1:1:1. Upper hexane layer was separated
and the absorbance was measured at 450 nm. Conjugated diene
was measured by adding 0.8 ml of ethanol to 0.2 ml of liposome
suspension and measuring the absorbance at 230 nm against eth-
anol as blank. Protection against oxidation was represented as pro-
tection factor calculated as follows,
Protection factor
X %increase in A230 of sample at time 
¼ %increase in A230 of control at time t =n
t
where, t is 2,4,6,8 and 24 h and n = 5.
2.4.3. Inhibition of singlet oxygen mediated oxidation
Singlet oxygen was generated by methylene blue photosensiti-
sation (Fukuzawa, Inokami, Tokumura, Terrao, & Suzuki, 1998).
Liposome suspension with samples was prepared as earlier, using
methylene blue solution (50 lM) instead of water. To initiate the
generation of singlet oxygen, the samples were kept under light
along with a control, and a-tocopherol was taken as positive con-
trol. The samples were drawn at intervals of 0, 2, 4, 6, 8 and 24 h for
determination of carotenoid and conjugated diene as explained
earlier. Protection factor was calculated using the percent increase
in conjugated diene.
2.5. Statistical analysis
Each experiment was carried out in triplicate and significant
differences between samples were analysed by ANOVA and mean
separation was accomplished by Duncan’s multiple range test
(Statsoft, 1999).
3. Results and discussion
3.1. In vitro antioxidant activities
Antioxidant activity of crude extract from shrimp waste and its
fractions were evaluated using various in vitro assays (Table 1).
DPPH-scavenging activity of crude extract and its fractions were
in the range of 3.08–3.74 mg TBHQ equivalent/mg of sample, the
highest activity being in fraction F3. However, there was no signif-
icant difference (p > 0.05) in DPPH-scavenging activity among dif-
ferent samples. ABTS radical-scavenging activity was significantly
(p < 0.05) higher (0.58 ± 0.8 mg TBHQ equivalent/mg extract) in
fraction F3 than for the crude extract and the other two fractions.
Hypochlorite ion scavenging activity was also higher in fraction F3,
followed by crude extract. Nitric oxide scavenging activity was
very low in fraction F1, and was highest in fraction F3, followed
by crude extract. Metal chelating and reducing activity was also
highest in fraction F3. However, singlet oxygen quenching activity
was higher in fraction F1 but was not significantly different from
that of fraction F3.
Fraction F3 was found to have the highest antioxidant activity,
while the activity of fraction F2 was similar to that of the crude ex-
tract. Column chromatographic separation employed in the study
preferentially elutes triglycerides and sterol esters in the hexane
fraction (F1). Carotenoid esters are eluted with hexane: acetone
(92:8) (F2) and free carotenoids are eluted with increased concen-
tration of acetone in the mobile phase (Lin et al., 2005). Astaxan-
thin contributed to 92% of total carotenoids in fraction 3, while
fraction 2 contained 69.7% astaxanthin esters (Table 2). Astaxan-
thin content in the crude extract was also high (66.7%). The total
 R. Sowmya, N.M. Sachindra / Food Chemistry 134 (2012) 308–314 311

Table 1
Antioxidant activity of crude extract (CE) and its fractions (F1–F3) as measured by different antioxidant assays.

CE F1 F2 F3
NS
DPPH scavenging (TBHQ equivalent mg/mg extract) 3.30 ± 0.42 3.08 ± 0.32 3.56 ± 0.26 3.74 ± 0.47
ABTS scavenging (TBHQ equivalent mg/mg extract) 0.30 ± 0.05a 0.32 ± 0.04a 0.40 ± 0.06a 0.58 ± 0.08b
Singlet oxygen quenching (a-tocopherol equivalent mg/mg extract) 2.10 ± 0.08a 3.93 ± 0.30b 2.17 ± 0.14a 3.88 ± 0.27b
Metal chelating (EDTA equivalent lg/mg extract) 21.1 ± 3.95a 26.3 ± 2.15ab 30.2 ± 2.79b 33.0 ± 5.89b
Reducing activity (ascorbic acid equivalent mg/mg extract) 0.54 ± 0.06a 0.70 ± 0.13a 1.07 ± 0.12b 1.52 ± 0.15c
Hypochlorite scavenging (gallic acid equivalent mg/mg extract) 0.14 ± 0.04a 0.08 ± 0.02b 0.11 ± 0.01ab 0.23 ± 0.02c
Nitric oxide scavenging (a-tocopherol equivalent mg/mg extract) 2.23 ± 0.23a 0.90 ± 0.07b 1.63 ± 0.32a 4.47 ± 0.55c

NS – not significant (p > 0.05); values in a row with different superscripts differ significantly (p < 0.05).

carotenoid content was also higher in fraction F3. Carotenoid con-
tent was negligible in fraction F1.
Different assays can be employed to determine the antioxida-
tive power of a compound (Singh & Singh, 2008). In the present
study, it was observed that the shrimp waste extract and its frac-
tions, particularly the fraction rich in astaxanthin, possess strong
antioxidant activity as detected in different assays. The results also
indicated that the crude extract and its fractions can act as both
preventive and chain-breaking antioxidants. Antioxidant activity
of astaxanthin is well established (Lawlor & O‘Brien 1995; Goto
et al., 2001). Most of the studies on evaluation of antioxidant activ-
ity of carotenoids used the pure carotenoids. Shrimp waste extract
contains other antioxidants, such as phenolics, in addition to
carotenoids (Seymour, Li, & Morrissey, 1996). Shellfish are also
known to contain several other lipophilic antioxidants, such as
tocopherol and ubiquinol (Passi, De, Puddu, & Littarru, 2002).
Antioxidants are known to have synergistic action (Milde, Elstner,
& Grassmann, 2007; Shixian et al., 2005). It is possible that compo-
nents other than carotenoids are also responsible for radical-
scavenging activity of extracts. However, as the activity correlated
with the carotenoid content of the extract, it can be presumed that
carotenoids are the major antioxidative principles in the shrimp
exoskeleton extract.
Singlet oxygen is implicated in many diseases. Hence, the sin-
glet oxygen quenching activity of natural compounds indicates
their usefulness as antioxidants. The best example of an antioxi-
dant action of carotenoids involves the ability of these pigments
to quench or inactivate excited singlet oxygen (Foote & Denny,
1968). The efficiency of singlet oxygen quenching of the carote-
noids has been shown to be related to the number of conjugated
double bonds (Conn, Schalch, & Truscott, 1991). Astaxanthin is
known to have strong singlet oxygen quenching activity (Nishida,
Yamashita, & Miki, 2007). As the shrimp extract used in the study
contains astaxanthin and its esters as major carotenoids, it exhibits
strong singlet oxygen quenching activity, which indicates its use-
fulness as a natural antioxidant for disease prevention.
NO is an important bioregulatory molecule, which has a num-
ber of physiological effects, including control of blood pressure,
neural signal transduction, platelet function, antimicrobial and
antitumour activity, and during infections and inflammations, for-
mation of NO is elevated and may bring about some undesired del-
eterious effects (Marcocci, Maguire, Droy-Lefaix, & Packer, 1994).
The products of NO, such as nitrate, nitrite and nitrosoamines,
are highly genotoxic, (Wink et al., 1991). Hence the NO scavenging
activity of the samples tested indicates their usefulness.
3.2. Inhibition of liposome oxidation
It is also important to study the behaviour of antioxidants in
membrane systems. One of the best, and oldest, model systems
to study the antioxidant action of carotenoids involves the use of
liposomes (Anderson & Krinsky, 1973). Liposomes have been used
to evaluate the behaviour of carotenoids in membrane model sys-
tems. Hence in the present study, investigations were carried out
to evaluate the antioxidant effect of crude carotenoids extract from
shrimp waste and its fractions with respect to inhibition of thermal
and singlet oxygen mediated oxidation of egg yolk PL liposomes.
PL liposomes with or without crude carotenoid extract or its
fractions were subjected to thermal oxidation at 50 °C and the
oxidation was monitored by increase in conjugated diene
content. a-Tocopherol was used as a positive control. In the case
of liposomes without any sample, the conjugated diene content in-
creased rapidly and at the end of 24 h there was a 23.7 ± 6.0-fold
increase compared to the level at time zero (Fig. 1A). Compared
to control, in the liposomes-containing samples, the increase was
marginal, with highest increase being 4.4 ± 1.7-fold in liposome
incorporated with fraction F1. Fraction F3 inhibited the oxidation
better than the known antioxidant a-tocopherol, as the increase
was only 1.4-fold with fraction 3 compared to 2.2-fold with a-
tocopherol. Even in the crude extract the increase was only 1.9-fold
at the end of 24 h.
In order to compare the inhibitory potential of different sam-
ples, a protection factor was calculated as explained in methods
section. The protection factor is an arbitrary unit which gives the
inhibitory effect compared to control. Fraction F3 showed signifi-
cantly (p < 0.05) higher protection against thermal oxidation of lip-
osomes with a protection factor of 22.6 ± 1.7 units (Fig. 1B). Even
the crude extract (12.0 ± 0.9 units) showed higher inhibitory activ-
ity than a-tocopherol (8.5 ± 1.5 units). Among all the fractions,
fraction F1 showed lowest (3.7 ± 0.4 units) inhibitory activity.
Carotenoids undergo degradation when they behave as antiox-
idants, as they prevent the oxidation of lipids, by making them-
selves available for reactions with radicals instead of lipids. In
some cases, as reported by Chen and Djuric (2001), carotenoids
in liposomes may be destroyed by the free radicals and are not
effective in preventing lipid peroxidation. However, in the present

Table 2
Total carotenoid content (as astaxanthin) and carotenoid profile (by HPLC) of crude extract and its fractions.

Sample Total carotenoid (lg astaxanthin/mg) Carotenoid profile (% of total carotenoid)

Astaxanthin Astaxanthin esters Other carotenoids
Crude extract 438 ± 21.4 66.7 ± 5.6 20.0 ± 3.2 13.3 ± 1.9
Fraction F1 – – – –
Fraction F2 384 ± 16.8 0.6 ± 0.2 69.7 ± 4.6 29.7 ± 3.4
Fraction F3 956 ± 32.6 92.0 ± 2.8 0.6 ± 0.1 7.4 ± 1.5
312 R. Sowmya, N.M. Sachindra / Food Chemistry 134 (2012) 308–314

A 35 A 7 Control
Control CE
30
CE 6 F1
Fold Increase in OD

25 F1
F2

Fold Increase in OD
F2 5
20 F3
F3
4 alpha-Tocopherol
15 alpha-Tocopherol

10 3
5
2
0
2 4 6 8 10 12 14 16 18 20 22 24 1
Time (hrs)
0
B 30 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs)
25 d
Protection Factor

B 20
20
18 c
15 16
a

Protection factor
c 14
10
c 12
5 b 10
8 a
0 ab
CE F1 F2 F3 alpha 6
ab
Tocopherol 4 b
2
Fig. 1. Increase in conjugated diene content (OD at 230 nm) during thermal
oxidation of liposomes (A) and protection factor crude extract and its fractions 0
along with tocopherol against thermal oxidation of liposomes (B) (Bars with CE F1 F2 F3 alpha-
different letters differ significantly, p < 0.05). Tocopherol

Fig. 3. Increase in conjugated diene content (OD at 230 nm) during singlet oxygen
mediated oxidation of liposomes (A) and protection factor of crude extract and its
study it was observed that in fraction F3 which showed higher
fraction along with tocopherol against singlet oxygen mediated oxidation of
antioxidant activity, even after 24 h, only 48% carotenoids were liposomes (Bars with different letters differ significantly, p < 0.05).
lost compared to almost 80% loss of carotenoids in fraction F2
(Fig. 2). This indicates that in addition to carotenoids, other compo-
nents are also involved in inhibition of thermal oxidation of containing fraction F3, the increase was marginal (1.7 ± 0.2-fold).
carotenoids. Fraction F3 inhibited the oxidation better than the known antiox-
PL liposomes with or without crude carotenoid extract or its idant a-tocopherol (2.2 ± 0.1-fold). Even in crude extract the in-
fractions were subjected to oxidation by singlet oxygen generated crease was lower (2.7 ± 0.3-fold) compared to fraction F1 and
by photo sensitisation of methylene blue, and the oxidation was fraction F2. The higher antioxidant activity of fraction F3 is indi-
monitored by increase in conjugated diene content. a-Tocopherol cated by a higher protection factor (14.1 ± 3.2 units) compared to
was used as a positive control. In case of liposomes without any a-tocopherol (6.2 ± 0.1 units). Comparatively, fraction F2 showed
sample, the conjugated diene content increased rapidly and at the lowest activity (2.7 ± 0.3 units) (Fig. 3B). The in vitro assays
the end of 24 h the increase was 5.1 ± 0.8-fold, compared to the le- for singlet oxygen quenching activity of extracts had indicated
vel at time zero (Fig. 3A). Compared to the control, in the liposomes (Table 1) higher activity in fraction F1, followed by fraction F3,

100 CE 100
F2 90
Residual carotenoid level (%)

90 CE
Residual carotenoid level (%)

80 F3 80 F2
70 70 F3
60 60
50 50
40 40

30 30

20 20

10 10

0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time (hrs) Time (hrs)

Fig. 2. Reduction in carotenoid content of liposomes incorporated with crude Fig. 4. Reduction in carotenoid content of liposomes incorporated with crude
extract (CE), astaxanthin ester rich fractions (F2) and astaxanthin rich fractions (F3) extract (CE), astaxanthin ester-rich fraction (F2) and astaxanthin-rich fraction (F3)
during thermal oxidation of liposome. during singlet oxygen mediated oxidation of liposomes.
 R. Sowmya, N.M. Sachindra / Food Chemistry 134 (2012) 308–314
fraction F2 and CE, which is different from the observations made
with liposome oxidation.
Unlike thermal oxidation, the carotenoid content in fraction F3
showed higher degradation with only 17.4 ± 2.5% residual caroten-
oid content at the end of 24 h of oxidation (Fig. 4). This indicates
that the inhibition of singlet oxygen mediated oxidation of lipo-
somes is mainly because of carotenoids, particularly astaxanthin,
as fraction F3 is rich in astaxanthin.
The antioxidant activity of carotenoids in liposomes is due to
their biophysical interaction with the lipid membrane (Woodall,
Britton, & Jackson, 1997). The factors that control antioxidant
capacity include the interactions of the carotenoid with the lipid bi-
layer and the resultant membrane modifications upon carotenoid
incorporation and the extent of carotenoid aggregation (Cantrell,
McGarvey, Truscott, Rancon, & Beohm, 2003). Carotenoids are the
effective modulators of physical properties of model and natural
membranes (Kosteka-Gugala, Latowski, & Strzalka, 2003). The anti-
oxidant activity of carotenoids in lipid membrane has also been
attributed to the modification of membrane permeability of radical
generators (Baros, Pinto, Colepicolo, & Pedersen, 2001). Carotenoids
with keto- and hydroxyl-groups on both ends of the molecule such
as astaxanthin strongly decrease water and small molecules perme-
ability across the lipid bilayer (Wisniewska & Subczynski, 1998).
Thus, in addition to a direct scavenging ability against reactive oxy-
gen species (ROS), some polar carotenoids also inhibit the penetra-
tion of oxidative substances and, consequently, the initiation of a
lipid peroxidation process. These factors may be responsible for
the difference in behaviour of the samples observed with respect
to singlet oxygen quenching by in vitro assay and in liposomes.
The reports available on antioxidant activity of shrimp carote-
noids are restricted to the activity of protein isolates containing
carotenoids, which is due to both carotenoids as well as hydrolysed
protein (Meenata et al., 2011; Sachindra & Bhaskar, 2008; Sowmya
et al., 2011). This study demonstrated the strong antioxidant activity
of carotenoid-containing solvent extract from shrimp exoskeleton.
4. Conclusion
Shrimp processing discards are an important source of carote-
noids, particularly that of astaxanthin and its esters. The crude
carotenoids extract obtained by solvent extraction of shrimp pro-
cessing discards, and its fractions were evaluated for their antiox-
idant activity by in vitro assays and in a membrane model system
using PL liposomes. The results indicated the strong antioxidant
activity of the crude extract and the astaxanthin-rich fractions ob-
tained from the crude extract. The antioxidant activity of shrimp
carotenoid extract indicates its potential for use as a natural anti-
oxidant for use in food and biomedical applications.
Acknowledgments
The authors wish to thank the Director, CFTRI for encourage-
ment and for the facilities. This study formed a part of the work
funded by Department of Biotechnology, Govt. of India.
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