The Open Conference Proceedings Journal, 2011, 2, 95-103
95
Open Access
Extraction, Characterization, Antioxidant and Anti-Inflammatory
Properties of Carotenoids from the Shell Waste of Arabian Red Shrimp
Aristeus alcocki, Ramadan 1938
S. Sindhua and P.M. Sherief*,b
a
Department of Processing Technology, College of Fisheries, Panangad. P.O, Kochi 682 506, Kerala, India
b
Department of Processing Technology, College of Fisheries, Panangad. P.O, Kochi 682 506, Kerala, India
Abstract: Shrimp processing waste is the single largest industrial waste in India causing diverse environmental problems.
Extraction of carotenoids from the shell waste of the Arabian red shrimp Aristeus alcocki was investigated using different
organic solvents, and vegetable oils, under wet and dry conditions, with and without deproteinization. The highest
carotenoid yield was obtained with non-deproteinized wet waste extracted using acetone. The carotenoid yield was found
to be double that of Pandalus borealis shell waste, which is currently used as one of the commercial sources of natural
astaxanthin. Thin Layer Chromatography (TLC) analysis of the carotenoid extract showed that it contains free
astaxanthin, astaxanthin monoester and astaxanthin diester in the ratio 1:1:2. gas chromatography (GC) analysis of the
fatty acids esterified with astaxanthin revealed that saturated fatty acids, monounsaturated fatty acids and poly unsaturated
fatty acids (PUFAs) are in the ratio 5:3:2 in monoester, whereas in diester they are in the ratio 4:3:3. The main fatty acids
in monoester and diester are palmitic acid, oleic acid, stearic acid and -3 PUFAs: docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA). The in vitro antioxidant activity of the carotenoid extract showed significant hydroxyl
radical scavenging activity, superoxide anion scavenging activity and inhibition of lipid peroxidation at nanogram levels.
The carotenoid extract significantly reduced carageenan induced paw edema in mice, percentage inhibition being 47.83
and 67.11 percent at astaxanthin concentrations of 0.5 mg/kg body weight and 1.0 mg/kg body weight, respectively. The
inhibition of inflammation at 1.0 mg/kg body weight was greater than that produced by the standard reference drug
diclofenac. The strong antioxidant and anti-inflammatory activities exhibited by the carotenoid extract of the shrimp shell
might be due to the combined action of astaxanthin and -3 PUFAs present in the astaxanthin esters.
Keywords: Anti-inflammatory, Antioxidant, Aristeus alcocki ,Astaxanthin, carotenoids, shell waste.
1. INTRODUCTION
Shrimp processing is one of the major food industries in
India. Shrimp processing waste is the single largest industrial
fish waste in the country causing diverse environmental
problems. Only 40% of the shrimp is edible and remaining
60 % account for the processing discards [1]. These discards
find very little practical application at present and are
categorized as a major environmental contaminant. Effective
utilization of the waste can resolve many of the
environmental concerns facing the shellfish processors [2].
In addition to the traditional uses, shrimp waste is one of the
important natural sources of carotenoid [3-4]. Shrimp
discards could be the cheapest raw material for carotenoid
recovery, and later could be a better alternative to synthetic
carotenoid [5]. The major component of carotenoids of
shrimp and crab shell backs were mono and diesters of
astaxanthin [2] a very potent antioxidant with some unique
properties suitable for use as a drug or food supplement.
Effective utilization of shrimp shell waste will enhance its
status as a biomedical research material, for development of
natural medicine without side effects.
*Address correspondence to this author at the Department of Processing
Technology, College of Fisheries, Panangad. P.O, Kochi 682 506, Kerala,
India; Tel: +91 484 2700274; Fax: +91 484 2700337;
E-mail: pmsherief@gmail.com
2210-2892/11
Several organic solvent systems were reported as
extraction solvent for the extraction of carotenoids from
different crustacean shell discards. Sachindra et al. [6] has
patented a method for extraction of carotenoids from shrimp
waste using solvent mixtures acetone & hexane and
isopropyl alcohol & hexane. Sachindra et al., [7] studied the
extractability of shrimp waste carotenoids using different
organic solvents and solvent mixtures to optimize the
extraction conditions for maximum yield. Organic solvents
such as acetone, ethanol, hexane can be used for the
extraction of carotenoids [8-12]. Methods are available for
extraction of carotenoids from crustacean wastes using
vegetable oils [13-20].
The present study is aimed at complete utilization of
shrimp waste or to “value add” the same. The novel
extraction techniques raise the possibility of not only
accessing a new source of astaxanthin but also finding an
environmentally friendly use of thousands of tonnes of
prawn waste discarded by seafood lovers around the world
each year. The effect of deproteinization on extraction yield
of carotenoids is also investigated in this study. The residue
available after extraction of carotenoids can be used for
production of chitin and chitosan, thus having an integrated
approach for efficient utilization of shrimp waste [21]. Thus the
generation of dual income from this cheap raw material will
lead to a profitable waste utilization strategy without causing
2011 Bentham Open
96 The Open Conference Proceedings Journal, 2011, Volume 2
Sindhu and Sherief
environmental pollution. The in vitro antioxidant property and
anti-inflammatory activity of shrimp shell astaxanthin were
also studied.
oil) with and without deproteinization. Waste was subjected
to deproteinization using alkali and enzyme as described in
section 2.3.
2. MATERIALS AND METHODS
A known weight of homogenized wet and dry shrimp
shell waste (1g) was extracted with 10 ml of solvent in order
to assess astaxanthin recovery. The carotenoid extract was
filtered using Whatman No.42 filter paper. Recovered
shrimp waste was repeatedly extracted with fresh solvent
until the filtrare was colourless, to a maximum of three
times. The pooled extract was collected for the quantification
of astaxanthin. In the case of acetone extraction the pooled
extract was collected in a separating funnel, 12.5ml of
petroleum ether (BP 40-600C) and 9.4ml of 0.73% (w/v)
NaCl solution were added. After thorough mixing the
epiphase was collected. To the lower phase an equal volume
of water was added, mixed and the epiphase was collected,
the pooled epiphase was evaporated to dryness under a
stream of nitrogen. The pooled extract in the case of
hexane:isopropanol (3:2, v/v) extraction was phase separated
with equal volume of 1% (w/v) NaCl solution. The epiphase
was collected and dehydrated with anhydrous sodium
sulphate, and then evaporated to dryness under vacuum and
the residue was dissolved in a 5 ml of hexane. In other cases
the solvent was removed under vacuum and redissolved in 5
ml of hexane. The ratio of oil: waste used in vegetable oil
extraction was 2:1 for wet sample and 4:1 for dry samples.
An antioxidant butyl hydroxy toluene (BHT) was added at
0.05% (w/v) and heated at 700C for 150 min, centrifuged and
the pigmented oil was recovered.
2.1. Preparation of Raw Material
Shell waste from the deep sea shrimp Aristeus alcocki
was collected from the processing plants: RF Exports Pvt.
Ltd, Chandiroor and Caps Seafoods Pvt. Ltd, Vypeen,
Kerala, India. The waste was transported to the laboratory in
an insulated box in iced condition. Aristeus alcocki is
processed as headless (HL) and peeled undeveined (PUD),
the major product styles marketed under the trade name “red
ring”. The product styles generate waste such as
cephalothorax, abdominal shell and tail portion. Adhering
meat from the cephalothorax was removed and the waste
was washed under running water and dried under shade.
They were packed in polyethylene bags and stored at -200C
until use. A second lot was stored wet, after removing the
adhering meat, was packed in polythene covers and stored at
-200C. Both wet and dry samples were homogenized in a
laboratory mixer (Crompton Greaves, India) prior to
extraction of carotenoids and estimation of different
components such as protein, chitin, lipids and ash in the
shell.
2.2. Proximate Composition
Moisture, ash, total nitrogen and total lipids were
quantified according to [22]. Chitin was determined
according to the method of Spinelli et al., [23]. Briefly,
samples were extracted with 2 % NaOH (w/v) followed by
demineralization with 5% HCl. The total protein content of
the waste was calculated by subtracting the chitin nitrogen
[24] from the total nitrogen, then using a conversion factor
of 6.25. All analyses were completed in triplicate. All the
chemicals used in the various tests were of either analar or
guaranteed reagent.
2.3. Deproteinization of Shrimp Shell Waste
Alkali deproteinization of shell waste was carried out
according to the method of Shahidi and Synowiecki [25].
Enzymatic deproteinization of shrimp waste was carried out
using enzyme pancraetin (from pig pancreas, Merck India
Ltd, Mumbai) according to the modified method of [19]. The
deproteinized residue was dried and total nitrogen of the
supernatant was determined by Microkjeldahl’s method [22].
Protein
content
was
calculated.
Percentage
of
deproteinization was calculated using the formula
% Deproteinization = Protein in supernatant x 100
Protein in waste
2.4. Extraction of Astaxanthin from Shrimp Shell Waste
Extraction of astaxanthin from shrimp shell waste was
investigated by application of the methods of Barbosa et al.
[26], Chen and Meyers, [15], Sachindra et al. [7], Kobayashi
et al. [27] and Sachindra and Mahendrakar [18] using
different organic solvents and vegetable oils. Homogenized
shrimp waste was extracted using four different organic
solvents [acetone, ether: acetone:water 15:75:10 (v/v/v),
hexane:isopropanol 3:2 (v/v), 90% acetone) and three
different vegetable oils, (coconut oil, sunflower oil, soyabean
2.5. Quantification of Astaxanthin
The organic solvent extracted astaxanthin was quantified
by measuring absorbance at 470 nm and using the equation
of Kelley and Harmon [28].
AST(μg / g) =
A x D x 10 6
100 x G x d x E1%
1cm
Where AST is astaxanthin concentration in g/g, A is
absorbance, D is volume of extract in hexane, 106 is dilution
multiple,G is weight of sample in g, d is cuvette width(1cm)
and E is extinction co-efficient, 2100.
For samples extracted in oil, quantification was done by
measuring absorbance at 485nm and using an extinction coefficient of 2155 in the above equation.
2.6. Identification of Different Components in Shrimp
Shell Waste Extract by thin Layer Chromatography
(TLC)
Analysis of different components in the shrimp shell
extract was done using thin layer chromatography (TLC)
based on the method of Kobayashi and Sakamoto [29]. For
this, a small volume of the extract was spotted on silicagel G
plate and developed using acetone: hexane 3:7 (v/v). The
separated bands were identified using standard astaxanthin
(Source: Green algae; Manufacturer: Sigma Chemicals,
USA) and internationally accepted Rf values for astaxanthin
monoester and astaxanthin diester. The different fractions,
astaxanthin, astaxanthin monoester, astaxanthin diester were
quantified by scraping out the respective bands in TLC plate.
The astaxanthin present in the scraped out sample was
Extraction, Characterization, Antioxidant
redissolved in 5 ml of hexane and quantified as described
earlier (2.5).
2.7. Analysis of Fatty Acids in Astaxanthin Monoester
and Astaxanthin Diester by Gas Liquid Chromatography
2.7.1. Saponification
The astaxanthin monoester and diester fractions
separated in TLC were redissolved in 2 ml of acetone and
the extract was transferred to a round bottom flask. Acetone
was evaporated under nitrogen. Added 10 ml of methanol
and 1 ml of 40 % NaOH and the flask was connected to an
air condenser, refluxed with nitrogen. The sample was
saponified by boiling for 30 minutes. After cooling the
unsaponifiable matter was removed by the addition of 10 ml
of petroleum ether (B.P 600C-800C). The aqueous layer was
acidified with 1 ml of concentrated hydrochloric acid (98%
HCl). Free fatty acid (FFA) was extracted with 5 ml of
petroleum ether (BP 400C-600C) and dried under anhydrous
sodium sulphate.
2.7.2. Gas Chromatography (GC)
The free fatty acids obtained from above were
quantitatively converted to fatty acid methyl esters (FAME)
using Boron triflouride-methanol reagent by the method of
Metcalfe et al. [30]. A known volume of the free fatty acid
extract in petroleum ether (5ml) was taken in a round bottom
flask and evaporated completely under nitrogen and 5 ml of
0.5 N methanolic NaOH was added. This mixture was
refluxed under water condenser (Borosil,India) for 5-10 min
under a nitrogen atmosphere. 6 ml of boron trifluoride methanol (BF3-CH3OH) solution was added using a glass
pipette through the condenser and continued boiling for 2
min. The flask was removed and cooled to warmness and 6
ml of fully saturated NaCl solution was added. The
stoppered flask was shaken vigourously for 15 min and the
upper phase was collected. The aqueous phase was extracted
with further two 30 ml portions of the petroleum ether (B.P.
60-800C). The combined extract was washed with three 20
ml portions of water, dried over anhydrous Na2SO4, filtered
through Whatman No.1 filter paper and evaporated using a
flash evaporator (BUCHI, Labortechnik AG, Flawil) under a
stream of nitrogen. Methyl esters of fatty acids were taken in
5ml of hexane and were separated by gas chromatography
(Trace GC Ultra, Thermo Electron Corporation) equipped
with Elite 225 (Perkin Elmer) capillary column and a flame
ionization detector in the presence of hydrogen and air.
Nitrogen at a flow rate 0.5ml/min was the carrier gas. Other
GC conditions were: injector temperature - 2500C;
temperature programme - 1100C-4 min 2.70C/min-2400C5min; Detector at 2750C. The fatty acids were identified and
quantified by external standard method using the fatty acid
standard mixture purchased from M/s. Suppleco. The output
of the GC was integrated using Thermocard software
(Thermo Electron Corporation, Italy) and individual fatty
acids were expressed as per cent
2.8. Assay of Antioxidant Activity
2.8.1. Superoxide Anion Scavenging Activity
Superoxide anion (O2.-) generated from the photo
reduction of riboflavin was detected by nitroblue tetrazolium
(NBT) reduction method of McCord and Fridovich [31]. The
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97
reaction mixture contained 6mM ethylenediaminetetraacetic
acid (EDTA) containing 3μg NaCN; 2μM riboflavin; 50μM
NBT; 67mM KH2PO4-Na2HPO4 buffer, (pH 7.8) and various
concentrations of astaxanthin (5 ng/ml to 50 ng/ml) in a final
volume of 3ml. The tubes were illuminated under
incandescent lamp for 15 min. The optical density (OD) at
530 nm was measured using UV/VIS Spectrophotometer
(Model V-530, Jasco make, Japan) before and after
illumination. The inhibition of superoxide radical was
determined by comparing the absorbance values of the
control with that of treatments. Quercetin (Sigma Chemicals,
USA )was used as standard.
% inhibition = OD of control - OD of test X 100
OD of control
2.8.2. Inhibition of Lipid Peroxidation
The lipid peroxidation induced by Fe 2+ - ascorbate
system in beef liver homogenate was estimated by
thiobarbituric acid reaction method of Ohkawa et al. [32].
The reaction mixture contained 0.1 ml of beef liver
homogenate (25 % w/v) in Tris-HCl buffer (20mM, pH 7.0);
30 mM KCl; 0.16 mM FeSO4(NH4)2SO4. 6 H2O; 0.06 mM
ascorbate and various concentrations of astaxanthin (5 ng/ml
to 50 ng/ml) in a final volume of 0.5 ml. The reaction
mixture was incubated at 370C for 1h. After the incubation
period, 0.4 ml was removed and treated with 0.2 ml of
sodium dodecyl sulphate (SDS) (8.1 %); 1.5 ml of 0.8 %
thiobarbituric acid and 1.5 ml of 20 % acetic acid (pH 3.5).
The total volume was made up to 4 ml with distilled water
and then kept in a water bath at 95-1000C for 1h. After
cooling 1 ml of distilled water and 5.0 ml of n-butanol and
pyridine mixture (15:1, v/v) were added to the reaction
mixture, shaken vigourously in an electric cyclomixer and
centrifuged at 4000 rpm for 10 min. The butanol-pyridine
layer was removed and absorbance at 532 nm was measured
using UV/VIS Spectrophotometer, , (Model V-530, Jasco
make, Japan). Inhibition of lipid peroxidation was
determined by comparing the OD of treatment with that of
control. Catechin (Sigma Chemicals, USA) was used as the
standard at a concentration ranging from 100μg/ml to
1000μg/ml.
2.8.3. Hydroxyl Radical Scavenging Activity
Hydroxyl free radicals degrade 2-deoxy ribose to form
thiobarbituric acid reactive substances (TBARS) [33].
Hydroxyl radical scavenging activity was determined by
studying the competition between deoxyribose and the
astaxanthin extract for the hydroxyl radical generated from
Fe2+-ascorbate-EDTA-H2O2 system (Fenton’s reaction). The
reaction mixture contained 2.8 mM deoxyribose; 0.1 mM
FeCl3; 20 mM KH2PO4-KOH buffer (pH 7.4); 0.1 mM
EDTA; 1.0 mM H2O2 ; 0.1 mM ascorbic acid and various
concentrations of astaxanthin (10 ng/ml to 100 ng/ml) in a
final volume of 1 ml. The TBARS formed was estimated by
reaction with thiobarbituric acid (TBA) to form pink
coloured complex [32]. The hydroxyl radical scavenging
activity was determined by comparing absorbance of control
with that of treatments at 532nm. Catechin (Sigma
Chemicals, USA)was used as standard at a concentration
ranging from 100μg/ml to 1000μg/ml.
98 The Open Conference Proceedings Journal, 2011, Volume 2
2.9. Assay of Antiinflammatory Activity
2.9.1. Animals
Antiinflammatory activity of astaxanthin was studied
using male Balb/c mice. Male Balb/c mice weighing 20-25 g
body weight, purchased from Small Animal Breeding
Centre, Kerala Agricultural University (KAU), Thrissur. The
animals were housed under hygienic conditions in
polypropylene cages under 12 hour light and dark cycle. All
procedures involving animal care and experiments were in
accordance with the guidelines of Committee for the Purpose
of Control and Supervision of Experiments on Animals
(CPCSEA), New Delhi, India and with the approval of
Institutional Animal Ethics Committee (IAEC).
Sindhu and Sherief
many other factors. Nair et al. [35] reported proximate
composition of prawn waste as 75-80 % moisture, 30-35 %
ash (dry basis), 35-40 % protein (dry basis), 15-20 % chitin
(dry basis) and 3-5 % fat (dry basis). The values observed in
the present study also correlates with the above values, and
also with the values reported by Madhavan and Nair [36].
Table 1.
Components
Fresh (Mean ± SD)
Dried (Mean ± SD)
Moisture
70.74 ± 0.56
14.57 ± 0.16
Ash
10.36 ± 1.13
29.40 ± 0.69
Chitin
9.80 ± 0.53
23.80 ± 0.80
Lipid
1.03 ± 0.11
2.06 ± 0.02
Protein
5.40 ± 0.28
29.71 ± 0.85
2.9.2. Carrageenan Induced Paw Edema
Carrageenan induced paw edema was used for
determining the acute anti-inflammatory activity of
astaxanthin. Animals were divided into four groups
containing six animals in each group. In all the groups,
inflammation was produced by injecting 0.025 ml of a 1%
(w./v) freshly prepared carrageenan (Source: Irish moss;
Manufacturer: Central Drug House, New Delhi) solution in
the right hind paw of the mice. One group with carrageenan
alone served as positive control. The second and third groups
were administered with astaxanthin extract at a concentration
of 0.5 mg/Kg and 1.0 mg/Kg body weight intraperitoneally
one hour prior to carrageenan injection. The fourth group
was administered with a standard reference drug diclofenac
(Sigma Chemicals, USA) 10 mg/Kg intraperitoneally. The
paw thickness was measured using vernier calipers before
and 3 h after carrageenan injection [34].
Increase in paw thickness was calculated using the
formula Pt-Po, where Pt is the thickness of paw at time ‘t’
(i.e. 3h after carrageenan injection) and Po is the paw
thickness at ‘0’ time. Percentage inhibition was calculated
using the formula (1-T/C) x 100 where C is the increase in
paw thickness of the control and T is that of the treatments.
2.10. Statistical Analysis
The statistical analyses of all the experiments were done
using the statistical package SPSS 15 for windows. The
analysis of variance technique was used to determine the
significant difference between different treatments at 5 %
level of significance. Experimental data were expressed as
mean ± SD.
3. RESULTS AND DISCUSSION
3.1. Proximate Composition of Shrimp Shell Waste
The proximate composition of fresh and dried shrimp
shell waste are presented in Table 1. The major components
in shrimp shell waste are protein, ash and chitin. Proximate
composition of shrimp shell waste varies with species and
Table 2.
Proximate Composition of Shrimp Shell Waste of
Aristeus alcocki, Ramadan, 1938 in Fresh and Dried
Conditions, Per Cent by Weight (n = 3)
3.2. Deproteinisation of Shrimp Shell Waste
Deproteinisation of shell waste using alkali (KOH) and
enzyme (Pancreatin) was carried out. Alkali deproteinsation
was more efficient than enzyme deproteinisation. Per cent
deproteinisation obtained with alkali and enzyme pancreatin
are presented in Table 2. Holanda and Netto [19] reported
that astaxanthin recovery from shrimp waste was enhanced
with enzymatic hydrolysis of shrimp shell waste. This
contradicts the result of the present study which showed that
deproteinisation by enzyme or alkali decreases the extraction
yield of carotenoids from shrimp shell waste. In this study, it
was observed that when hydrolyzed astaxanthin leached into
the medium, decreasing the carotenoid content of the
residual shrimp waste.
3.3. Extraction of Carotenoids From Shrimp Shell Waste
The raw material used for the present study was shrimp
shell wastes of deep sea shrimp Aristeus alcocki commonly
called as Scarlet shrimp or Arabian red shrimp. To ensure
maximum yield of carotenoid pigments this deep sea species
was selected. The carotenoid astaxanthin was quantified in
the shrimp shell extract of Aristeus alcocki in the present
study at 470 nm in hexane. Cianci et al. [37] has reported
that astaxanthin has a typical absorption maxima at 472 nm
in hexane. The total carotenoids extracted from shrimp waste
of Xiphopenaeus kroyeri were refered as astaxanthin by
Holanda and Netto, [19].
Analysis of variance (Table 3) for extraction yields of
carotenoids in different extraction media, with different
samples demonstrated that the yield of carotenoids varied
significantly between extraction solvents, between wet and
dry samples and between deproteinised and non
Extent of Deproteinisation of Aristeus alcockii Shrimp Waste Using Enzyme and Alkali, Percentage of Total Protein n=3
Samples
Alkali deproteinization (%) (Mean ± SD)
Enzyme deproteinization (%) (Mean ± SD)
Wet
77.68 ± 0.31
66.61 ± 0.21
Dry
51.53 ± 0.25
35.34 ± 0.04
Extraction, Characterization, Antioxidant
Table 3.
The Open Conference Proceedings Journal, 2011, Volume 2
99
ANOVA for Extraction Yields of Carotenoids for Different Samples and Different Extraction Media
Source of variation
Sum of squares
Degrees of freedom
Mean sum of squares
F-value
Samples
35309.32
5
7061.86
6035.25*
Extraction medium
8054.48
6
1342.41
1147.26*
Interaction
13206.78
30
440.23
376.22*
Error
147.43
126
1.170
Total
56718.01
167
*Significant at 5 % level Critical difference:0.284.
deproteinised samples. The mean extraction yields of
carotenoids from shrimp shell waste with and without
deproteinisation in dry and wet condition are presented in
Table 4. The highest carotenoid yield of 87.14 ± 4.55 μg/g
was obtained with wet non deproteinised waste using
acetone. Sachindra et al. [7] reported that a 50:50 mixture of
isopropyl alcohol and hexane gave the highest yield
(43.91μg/g wet waste) of carotenoid compared to acetone
(40.60 μg/g wet waste). On the contrary in the present study
highest carotenoid yield was obtained with acetone (87.14
μg/g), when compared to 60:40 solution of hexane :
isopropanol that yielded (70.27μg/g). This difference may be
due to difference in species or due to high polarity of
acetone. Vimla and Paul [38] have reported that maximum
yield of carotenoids from Penaeus monodon waste was
obtained with acetone, compared to other solvents used for
extraction. Polar solvents are generally a good extraction
media for xanthophylls whereas non polar solvents are not
recommended as their penetration through the hydrophobic
mass that surrounds the pigment is limited [39].
The carotenoid yields from dry samples were
significantly reduced compared to that of wet samples. On
drying the shell colour was found to be bleached and the
yield of astaxanthin was found to be very low from dried
samples. Similarly the carotenoid yields from deproteinised
samples were significantly lower than those of non
Several reports are available on the yield of astaxanthin
from different species of deep sea shrimps. Of these, highest
yield of 14.8 mg/100 g dry waste was reported by Shahidi
and Synowiecki [25] from the waste of P. borealis shrimp.
In the present study we obtained an yield of 87.14 μg/g wet
waste from Aristeus alcocki. This is equivalent to 25.44
mg/100 g, on dry waste. The present study thus reveals that,
of the different species of deep sea shrimps Aristeus alcocki
shell waste is an excellent source of astaxanthin.
At present P. borealis shrimp waste is commercially
exploited for the production of natural astaxanthin (M/s
Bioprawns, Norway). Since astaxanthin content in Aristeus
alcocki shell waste is double the content of astaxanthin in P.
borealis there is ample scope for exploiting Aristeus alcocki
Acetone (μg/g)
Hexane:
Isopropanol
(μg/g)
90% acetone (μg/g)
Coconut oil (μg/g)
Soy bean oil (μg/g)
Sunflower oil (μg/g)
Extraction Yields of Carotenoids in Different Solvents and Vegetable Oils
Ether :acetone:
water (μg/g)
Table 4.
deproteinised samples. Oil extraction of carotenoids was
carried out using coconut oil, soybean and sunflower oil.
Although carotenoid yield was significantly lower when oil
was used as the extraction medium, of the three oils, coconut
oil gave the highest yield (3.32 ± 0.23 μg/g). Holanda and
Netto [19] reported that astaxanthin recovery from shrimp
waste using a mixture of solvents is more efficient than oil
extraction. This is in agreement with the present study. Also
it was observed that for any dry sample (deproteinized or
non deproteinized), 90 % acetone was found to be the best
solvent for carotenoid extraction.
Dry(D)
21.25 ± 0.86a,g
15.74±0.83b,g
20.34±0.35c,g
22.79±1.33d,g
3.32±0.23c,g
1.36±0.15f,g
1.48±0.20f,g
EDD
0.47±0.86a,h
1.15±0.19a,h
0.29±0.05a,h
1.17± 0.13a,h
ND
0.65±0.30a,c,g
0.43±0.04c,h
ADD
0.36±0.04a,h
0.74±0.08a.h
0.19±0.06a,h
0.75± 1.00a,h
0.22±0.03a,h
0.31±0.02a,h
0.37±0.07a,h
Wet(W)
40.22±1.15a,i
87.14±4.55b,i
70.27±2.58c,i
41.46±2.51d,i
23.05±2.09e,i
20.27±0.88f,i
18.06±0.82g,i
EDW
10.56±0.23a.j
21.43±1.33b,j
12.04±0.99b,j
1.10± 0.18c,h
5.07±0.17d,j
3.79±0.39e,j
4.01±0.61f,j
ADW
9.70±0.75a,k
17.38±0.59b,k
9.84±0.72b,k
1.36± 0.31c,h
4.51±0.36d,j
2.88±0.13d,k
2.40±0.21e,k
Extraction
Media
Raw
material
Values are mean ± SD of four different estimations. Values having the same superscript in the same column and same row are not significantly different at 5 % level.
D – Dry non deproteinized sample
W - Wet non deproteinized sample.
EDD – Enzyme deproteinized dry sample
EDW - Enzyme deproteinized wet sample.
ADD - Alkali deproteinized dry sample
ADW - Alkali deproteinized wet sample.
100 The Open Conference Proceedings Journal, 2011, Volume 2
Sindhu and Sherief
shell waste for the commercial production of natural
astaxanthin.
were palmitic acid (20.47%), oleic acid (18.19%) and
PUFAs: EPA (8.79%) and DHA (11.36%).
3.4. Determination of Different Components in Shrimp
Shell Waste Extract by Thin Layer Chromatography
The fatty acid profiles are in agreement with the findings
of Maoka and Akimoto [11], who reported that saturated
fatty acids constitute 45.55%, monounsaturated fatty acids
constitute 30.25% and PUFAs 29.3 % in the two monoester
fractions of spiny lobster Panulirus japonicus. Whereas in
astaxanthin diester saturated fatty acids, monounsaturated
fatty acids and PUFAs constituted 47.95%, 22.80% and
22.95% respectively. Crustaceans accumulate carotenoid in
both free and esterified forms and esterification increased the
stability of carotenoid in the lipid matrix. Maoka and
Akimoto [11] reported that 66 % of carotenoids were
esterified in spiny lobster Panulirus japonicus. The major
fatty acids esterified with astaxanthin were identified as
stearic acid, oleic acid and palmitoelic acid followed by
PUFAs such
as docosahexaenoic
acid
(DHA),
eicosapentaenoic acid (EPA). Similar results were obtained
in the present study with palmitic acid, oleic acid and stearic
acid being the major fatty acids in mono and diester followed
by PUFAs, DHA and EPA. Sachindra et al. [42] reported
that unsaturated fatty acid constituted 60.5% of the
carotenoid extract from the head of A. alcocki, while
saturated fatty acids (83.5 %) were predominant in the
carotenoid extract from the carapace of S. indica. The
present study thus reveals that the carotenoid extract from
Aristeus alcocki shell waste is mainly composed of free
astaxanthin, astaxanthin monoester and astaxanthin diester in
the proportion 1:1:2. The fatty acid composition of the
monoester reveals that saturated fatty acids, MUFA and
PUFA are in the ratio 5:3:2 whereas in diester they are in the
ratio 4:3:3.
Thin layer chromatographic separation of carotenoid
extracts from Aristeus alcocki yielded three distinct bands
(Table 5). The Rf values for the three bands were
respectively 0.33, 0.60, 0.78 which corresponded to
astaxanthin, astaxanthin monoester, astaxanthin diester. The
Rf values obtained for astaxanthin monoester and astaxanthin
diester are in agreement with the results reported by
Kobayashi and Sakamoto [29], 0.60 for astaxanthin
monoester and 0.75-0.85 for astaxanthin diester. The Rf
value of astaxanthin obtained in the present study is in
accordance with the Rf value obtained for the standard
astaxanthin and also the Rf value reported by Todd [40].
Spectrophotometric quantification of three bands showed
that the extract contained astaxanthin: astaxanthin
monoester: astaxanthin diester in the ratio 1:1:2 showing the
predominance of astaxanthin diester.
Sahidi et al. [3] and Sachindra et al. [5] have reported
that astaxanthin and its esters are the major carotenoids in
the marine crustaceans. Breithaupt [41] observed that
homogenous diester astaxanthin was the predominant
compound, followed by mixed diester astaxanthin in the
carotenoid extract from Pandalus borealis. Sachindra et al.
[42] reported that astaxanthin and its mono and diesters
(63.5-92.2%) were the major carotenoids in Aristeus alcocki
and Solonocera indica, two important deep sea species from
Indian waters. A quantitative study of carotenoid distribution
in those species have revealed a higher proportion of
esterified astaxanthin than the free form. A. alcocki had a
higher proportion of astaxanthin esters (61.7-70.8%)
compared to S. indica (43.8-58.4%).
Table 5.
Rf Value of Different Carotenoids in the Extract
from
Aristeus
alcocki,
(Mean
of
Three
Determinations)
Table 6.
Fatty Acid Composition of Astaxanthin Monoester,
as Per Cent of Fatty Acid
Sl. No.
Component
Area %
1
C12
3.002
2
C14
4.161
Carotenoid
Rf value
3
C16
18.382
Astaxanthin diester
0.78
4
C16:1
2.930
Astaxanthin monoester
0.60
5
C17
0.910
Astaxanthin
0.33
6
C18
9.281
7
C18:1
14.400
3.5. Fatty Acid Composition in Astaxanthin Monoester
and Astaxanthin Diester
8
C18:2
1.236
9
C18:3
1.867
The fatty acid composition in astaxanthin monoester and
astaxanthin diester are presented in Tables 6 and 7,
respectively. Astaxanthin monoester contained 49.29%
saturated fatty acids, 30.43 % monounsaturated fatty acids
and 20.28 % polyunsaturated fatty acids (PUFAs).
Astaxanthin diester contained 41.94 % saturated fatty acids,
29.91 % monounsaturated fatty acids and 29.85% PUFAs. In
the case of monoester, the main fatty acids esterified with
astaxanthin were palmitic acid (18.38%) and oleic acid
(14.40%). The main PUFAs present in the monoester were
eicosapentaenoic acid (EPA, 20:5, -3) (4.83%) and
docosahexaenoic acid (DHA, 22:6, -3) (6.58%). In the case
of diester, the main fatty acids esterified with astaxanthin
10
C20:1
8.468
11
C20:4
2.844
12
C20:3&C21
2.922
13
C20:5
4.832
14
C22
7.388
15
C22:1
1.758
16
C22:6
6.577
17
C24
6.165
18
C24:1
2.878
Extraction, Characterization, Antioxidant
Table 8.
The Open Conference Proceedings Journal, 2011, Volume 2
In Vitro Antioxidant Activity of Astaxanthin from Shrimp Shell Waste (Aristeus alcocki), IC50, n = 6
Activity
Astaxanthin (Mean ± SD)
Quercetin (Mean ± SD)
Catechin (Mean ± SD)
Superoxide radical scavenging activity
27.91 ± 0.54 ng/ml
41.21 ± 0.76 μg/ml
-
Inhibition of lipid peroxidation
26.54 ± 0.42 ng/ml
-
432 ±10.2 μg/ml
Hydroxyl radical scavenging activity
56.43 ± 1.06 ng/ml
-
842 ±16 μg/ml
Table 7.
101
Fatty Acid Composition of Astaxanthin Diester, as
Per Cent of Fatty Acid
Sl. No.
Component
Area %
1
C14
2.121
2
C16
20.472
3
C16:1
4.249
4
C17
0.947
5
C18
8.800
6
C18:1
18.197
7
C18:2
2.232
8
C18:3
1.116
9
C20:1
6.333
10
C20:4
3.988
11
C20:3&C21
1.690
12
C20:5
8.797
13
C22
3.201
14
C22:2
0.665
15
C22:6
11.360
16
C24
4.708
17
C24:1
1.125
overproduction occurs, resulting in oxidative stress. This
stress and the resultant damage have been implicated in
many diseases and a wealth of preventive drugs and
treatments are currently being studied. Thus, astaxanthin
exhibiting multiple antioxidant activity will find utility in
applications like antioxidant therapy, which is based on
reducing oxidative stress in the target tissues. Since synthetic
astaxanthin is a mixture of three stereoisomers (3R,3’R; 3S
3’S; 3R,3’S) astaxanthin from natural sources is preferred
for using it as an antioxidant. Astaxanthin from natural
sources is abundant in the isomer showing highest biological
activity (3R,3’R; 3S 3’S).
Kamath et al. [45] has reported that the IC50 values for
free radical scavenging activity of Haematococcus pluvialis
astaxanthin esters in vitro were 8.0 μg /ml. In the present
study the in vitro IC50 values reported for antioxidant activity
of astaxanthin from Aristeus alcocki shell waste are in the
range of ng/ml. This clearly indicates the astaxanthin extract
from Aristeus alcocki is a more powerful antioxidant than
the astaxanthin esters present in the Haematococcus
pluvialis. This may be due to a higher proportion of
astaxanthin diester and a higher content of poly unsaturated
fatty acids (20 % PUFAs in monoester and 30 % PUFAs in
diester) in the carotenoid extract obtained from Aristeus
alcocki shell. Thus, the powerful antioxidant property of
carotenoid extract of Aristeus alcocki may be attributed to
the antioxidant synergism of astaxanthin and poly
unsaturated fatty acids (PUFAs) present in the extract.
3.6. Antioxidant Activity
3.7. Antiinflammatory Activity
Astaxanthin extracted from shell waste of Aristeus
alcocki possessed significant hydroxyl radical scavenging
activity, lipid peroxidation-inhibiting activities and
superoxide radical-scavenging activity (Table 8). The extract
showed 50% inhibition (50 % inhibiting concentration) at
concentrations 56.43 ± 1.06 ng/ml, 26.54 ± 0.42 ng/ml,
27.91 ± 0.54 ng/ml. The standard antioxidants quercetin and
catechin showed antioxidant activity at microgram levels
whereas astaxanthin present in shrimp shell extract showed
in vitro antioxidant activity at nanogram levels. This clearly
indicates the high antioxidant potential of astaxanthin
extracted from Aristeus alcocki shell waste.
Carotenoid extract from Aristeus alcocki significantly
inhibited the acute inflammation induced by carageenan.
Analysis of variance for increase in paw thickness of Balb/c
mice with different treatments showed that the extract from
shrimp shell waste significantly reduced carageenan induced
paw edema. The reduction in edema was noted in a dose
dependent manner (Table 9). Astaxanthin concentrations at
0.5 mg/kg body weight and 1.0 mg/kg body weight inhibited
the inflammation by 47.83 and 67.11 percent (Table 9). The
inhibition of inflammation at 1.0mg/kg body weight was
greater than the standard reference drug diclofenac.
Bell et al. [43] in a feeding study with salmon showed
that the antioxidant synergism of vitamin E and astaxanthin
reduced malondialdehyde formation in an in vitro
stimulation of microsomal lipid peroxidation. Oxygen
derived free radicals or reactive oxygen species (ROS)
formed in the body during energy producing metabolic
process, play an important role in pathophysiology of a
number of diseases [44]. Normally oxygen free radicals are
neutralised by natural antioxidants. However, ROS become a
problem when either a decrease in their removal or their
Results of the present investigations reveal that
astaxanthin exhibited significant dose dependent antiinflammatory activity in acute inflammations, within mice in
a dose dependent manner. This confirms the findings of
previous studies. Ohgami et al. [46] demonstrated a dose
dependent anti-inflammatory effect of astaxanthin by
supression of nitric oxide (NO), prostaglandin E2 (PGE2)
and tumour necrosis factor (TNF-) production by directly
blocking nitric oxide synthase (NOS) enzyme activity.
Kurashige et al. [47] also reported that carrageenan induced
swelling of the paw of rats fed with astaxanthin was
102 The Open Conference Proceedings Journal, 2011, Volume 2
Table 9.
Sindhu and Sherief
Effect of Astaxanthin Extract on Carageenan Induced Paw Edema
Treatment
Initial paw thickness (mm)
Paw thickness on 3 h (mm)
Increase in paw thickness (mm)
% inhibition
Control
15.00 ± 0.09
25.37 ± 0.31
10.37 ± 0.13a
-
b
52.94
Standard (Diclofenac)
15.66 ± 0.08
20.54 ± 0.24
4.88 ± 0.26
0.5mg astaxanthin/kg body
weight
15.55 ± 0.11
20.96 ± 0.29
5.41 ± 0.18c
47.83
15.76 ± 0.09
19.17 ± 0.14
3.41 ± 0.16d
67.11
1.0mg astaxanthin
/kg body weight
Values expressed as mean ± S.D, n=6 animals, Values having the same superscript in same column are not significantly different at 5 % level.
significantly lower than that of control. This explains the
anti-inflammatory role of astaxanthin. Bennedsen et al. [48]
and Wang et al. [49] reported that dietary astaxanthin was
found to help fight symptoms of ulcer disease from
Helicobacter pylori which causes inflammation of gastric
tissues. Kim et al. [50] reported that astaxanthin is effective
in protection against gastric lesions induced by the use of
non steroid anti-inflammatory drugs such as naxopen.
Mahmoud et al. [51] reported that suppression of T-cell
activation makes astaxanthin as effective as commonly used
antihistamines and hence may have a role in novel
antiasthmatic formulations.
Astaxanthin has been studied extensively due to its
superior antioxidant and anti-inflammatory properties. The
present study demonstrates that natural astaxanthin from
Aristeus alcocki shrimp waste inhibits carrageenan induced
inflammatory response in mice. This anti-inflammatory
effects of astaxanthin from Aristeus alcocki shell has
important implications for the development of antiinflammatory drugs from shrimp shell waste.
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Accepted: May 19, 2011
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