INTRODUCTION

Astaxanthin is a ketocarotenoid that belongs to class of

phytochemicals known as tetraterpenoids, which are built from
eight isoprenes,C5H8 molecules; isopentenyl diphosphate (or IPP)
and dimethylallyl diphosphate (or DMAPP) as the initial precursor.
The colour is due to the extended chain of conjugated (alternating
double and single) double bonds at the centre of the compound.
This chain of conjugated double bonds is also responsible for the
antioxidant properties of astaxanthin (as well as other carotenoids)
as it results in a region of decentralized electrons that can be
donated to reduce a reactive oxidizing molecule.

Astaxanthin is found in microalgae, yeast, salmon, trout,

krill, shrimp, crayfish, crustaceans, and the feathers of some
birds. The red color of salmon meat and cooked shellfish is due to
this pigment. Professor Basil Weedon's group was the first to prove
the structure of astaxanthin by synthesis, in 1975. Astaxanthin
unlike some other carotenoids, is not converted to vitamin A
(retinol) in the human body. Too much vitamin A is toxic for a
human, animal, but astaxanthin has lower toxicity. It is antioxidant
with slightly lower antioxidant activity than other carotenoids.
Astaxanthin is a fat soluble compound, with increased absorption
when consumed with dietary oils. Astaxanthin was shown to
significantly influence immune function in several in vitro and in
vivo assays.

1
 The recovery of these valuable components from the waste
would not only improve the economy for crustacean processors, but
also would minimize the pollution potential of the Crustacean
waste. Crustacean waste could be the cheapest raw materials for
carotenoid recovery, and later could be a better and cheaper
alternative to synthetic carotenoid in aquaculture feed formulations
and in surimi based products. The U.S. Food and Drug
Administration (FDA) has approved astaxanthin as a food coloring
(or color additive) for specific uses in animal and fish foods 6. The
European Commission considers it food dye.

Marine fisher resourses are living natural resourses which

are self renewable with dynamic habitat. In India, the natural
resources are highly rich where annual harvestable fishery potential
in the country is estimated to increase in millions of tones day by
day. The marine crabs and shrimps were present in the landing
centers of Visakhapatnam and processing in sea food factories. In
recent days due to increase in consuming of sea food there is a
increase in production of processing food.

In the sea food processing centers and marketing places

there is a large scale stacking of shell waste. The marine waste
causes undesirable changes during the contamination starting post
harvesting period. This waste was dumped into surrounding
environment. In general the crabs and shrimps containing
carotenoid compound in their shells. These carotenoid have an anti
oxidant and anti microbial property.

2
 To minimize the pollution the crustacean waste can be
served as the cheap raw material for production of carotenoids. The
recovery of these valuable components from the waste would not
only improve the economy for crustacean processors, but also
would minimize the pollution potential of the Crustacean waste.
Crustacean waste could also be a better and cheaper alternative to
synthetic carotenoid in aquaculture feed formulations and in surimi
based products.

so my aim of the present study is extraction, identification,

antimicrobial and phytochemical analysis of astaxanthin from
shrimp shells collected from processing plants around
Visakhapatnam city.

3
 Review of Literature
Structure of Astaxanthin
Astaxanthin is a member of the xanthophylls, because it
contains not only carbon and hydrogen but also oxygen atoms.
Astaxanthin consists of two terminal rings joined by a polyene
chain. This molecule has two asymmetric carbons located at the 3,
3′ positions of the β-ionone ring with hydroxyl group (-OH) on
either end of the molecule. In case one, hydroxyl group reacts
with a fatty acid then it forms mono-ester, whereas when both
hydroxyl groups are reacted with fatty acids the result is termed a
di-ester.
Astaxanthin exists in stereoisomers, geometric isomers,
free and esterified forms. All of these forms are found in natural
sources. The stereoisomers (3S, 3′S) and (3R 3′R) are the most
abundant in nature. Haematococcus biosynthesizes the (3S, 3′S)-
isomer whereas yeast Xanthophyllomyces dendrorhous produces
(3R, 3′R)-isomer [Hussein et al.,2006]. Synthetic astaxanthin
comprises isomers of (3S, 3′S) (3R, 3′S) and (3R, 3′R). The
primary stereoisomer of astaxanthin found in the Antarctic krill
Euphausia superba is 3R, 3′R which contains mainly esterified
form, whereas in wild Atlantic salmon it is 3S, 3′S which occurs
as the free form. Astaxanthin has the molecular formulaC40H52O4.
Its molar mass is 596.84 g/mol.

4
 Figure 1. Planner structure of astaxanthin.

Astaxanthin is classified as a xanthophyll (originally

derived from a word meaning "yellow leaves" since yellow plant
leaf pigments were the first recognized of the xanthophyll family
of carotenoids), but currently employed to describe carotenoid
compounds that have oxygen-containing moities, hydroxyl (-OH)
or ketone (C=O), such as zeaxanthin and canthaxanthin. Indeed,
Astaxanthin is a metabolite of zeaxanthin and/or canthaxanthin,
containing both hydroxyl and ketone functional groups. Like
many carotenoids, astaxanthin is a colorful, lipid-soluble
pigment.
The recovery of these valuable components from the waste
would not only improve the economy for crustacean processors,
but also would minimize the pollution potential of the Crustacean
waste. Crustacean waste could be the cheapest raw materials for
carotenoid recovery, and later could be a better and cheaper
alternative to synthetic carotenoid in aquaculture feed

5
formulations and in surimi based products. The U.S. Food and
Drug Administration (FDA) has approved astaxanthin as a food
coloring (or color additive) for specific uses in animal and fish
foods (Sarada et al.,2012)

The European Commission considers it food dye and it is

given the E number E161j. Natural astaxanthin is considered
generally recognized as safe (GRAS) by FDA.

Source of Astaxanthin
The natural sources of astaxanthin are algae, yeast, salmon,
trout, krill, shrimp and crayfish. Astaxanthin from various
microorganism sources are presented in Table 1. The commercial
astaxanthin is mainly from Phaffia yeast, Haematococcus and
through chemical synthesis. Haematococcus pluvialis is one of
the best sources of natural astaxanthin. Astaxanthin content in
wild and farmed salmonids . Among the wild salmonids, the
maximum astaxanthin content in wild Oncorhynchus species was
reported in the range of 26–38 mg/kg flesh in sockeye salmon
whereas low astaxanthin content was reported in chum.
Astaxanthin content in farmed Atlantic salmon was reported
as 6–8 mg/kg flesh. Astaxanthin is available in the European (6
mg/kg flesh) and Japanese market (25 mg/kg flesh) from large
trout. Shrimp, crab and salmon can serve as dietary sources of
astaxanthin. Wild caught salmon is a good source of astaxanthin.
In order to get 3.6 mg of astaxanthin one can eat 165 grams of
salmon per day. Astaxanthin supplement at 3.6 mg per day can be
beneficial to health as reported by Iwamoto et al.

6
 Table 1: Microorganism sources of astaxanthin
Astaxanthin
(%) on the Dry
Sources Weight Basis Reference
Chlorophyceae
Haematococcus pluvialis 3.8 [17,18]
Haematococcus pluvialis (K-0084) 3.8 [22]
Haematococcus pluvialis (Local 3.6 [23]
isolation)
Haematococcus pluvialis 3.4 [24]
(AQSE002)
Haematococcus pluvialis (K-0084) 2.7 [25]
Chlorococcum 0.2 [26,27]
Chlorella zofingiensis 0.001 [28]
Neochloris wimmeri 0.6 [29]
Ulvophyceae
Enteromorpha intestinalis 0.02 [30]
Ulva lactuca 0.01 [30]
Florideophyceae
Catenella repens 0.02 [30]
Alphaproteobacteria
Agrobacterium aurantiacum 0.01 [31]
Paracoccus carotinifaciens (NITE 2.2 [32]
SD 00017)
Tremellomycetes
Xanthophyllomyces dendrorhous 0.5 [33]
(JH)
Xanthophyllomyces dendrorhous 0.5 [34]
(VKPM Y2476)
Labyrinthulomycetes

Thraustochytrium sp. CHN-3 0.2 [35]

(FERM P-18556)
Malacostraca
Pandalus borealis 0.12 [20]
Pandalus clarkia 0.015 [36]

7
 Figure. 2 Sourses of astaxanthin

Biochemistry of Astaxanthin
Astaxanthin contains conjugated double bonds, hydroxyl and
keto groups. It has both lipophilic and hydrophilic properties. The red
color is due to the conjugated double bonds at the center of the
compound. This type of conjugated double bond acts as a strong
antioxidant by donating the electrons and reacting with free radicals to
convert them to be more stable product and terminate free radical chain
reaction in a wide variety of living organisms. Astaxanthin showed
better biological activity than other antioxidants, because it could link
with cell membrane from inside to outside.

Figure. 3 3D structure of astaanthin

8
 Figure 4. Superior position of astaxanthin in the cell membrane

Bioavailability of Astaxanthin

Dietary oils may enhance the absorption of astaxanthin.

Astaxanthin with combination of fish oil promoted
hypolipidemic/hypocholesterolemic effects in plasma and its increased
phagocytic activity of activated neutrophils when compared with
astaxanthin and fish oil alone. Astaxanthin was superior to fish oil in
particular by improving immune response and lowering the risk of
vascular and infectious diseases.
The proliferation activity of T- and B-lymphocytes was
diminished followed by lower levels of O2, H2O2 and NO production,
increased antioxidant enzymes superoxide dismutase, catalase and
glutathione peroxidase (GPx), and calcium release in cytosol after
administration of astaxanthin with fish oil. Bioavailability and

9
antioxidant properties of astaxanthin were enhanced in rat plasma and
liver tissues after administration of Haematococcus biomass dispersed in
olive oil. Astaxanthin is a fat soluble compound, with increased
absorption when consumed with dietary oils. Astaxanthin was shown to
significantly influence immune function in several in vitro and in vivo
assays. Lipophilic compounds such as astaxanthin are usually
transformed metabolically before they are excreted, and metabolites of
astaxanthin have been detected in various rat tissues.
Astaxanthin bioavailability in human plasma was confirmed with
single dosage of 100 mg. Its accumulation in humans was found after
administration of Haematococcus biomass as source of astaxanthin.
Astaxanthin bioavailability in humans was enhanced by lipid based
formulations; high amounts of carotenes solubilized into the oil phase of
the food matrix can lead to greater bioavailability. A recent study
reported that astaxanthin accumulation in rat plasma and liver was
observed after feeding of Haematococcus biomass as source of
astaxanthin.

Biological Activities of Astaxanthin and Its Health Benefits

1. Antioxidant Effects:
An antioxidant is a molecule which can inhibit oxidation.
Oxidative damage is initiated by free radicals and reactive oxygen
species (ROS). These molecules have very high reactivity and are
produced by normal aerobic metabolism in organisms. Excess oxidative
molecules may react with proteins, lipids and DNA through chain
reaction, to cause protein & lipid oxidation and DNA damage which are
associated with various disorders. This type of oxidative molecules can

10
be inhibited by exo and endogenous antioxidants such as carotenoids.
Carotenoids contain polyene chain, long conjugated double bonds,
which carry out antioxidant activities by quenching singlet oxygen and
scavenging radicals to terminate chain reactions. The biological benefits
of carotenoids may be due to their antioxidant properties attributed to
their physical and chemical interactions with cell membranes.
Astaxanthin had higher antioxidant activity when compared to various
carotenoids such as lutein, lycopene, α-carotene and β-carotene reported
by Naguib et al.
The antioxidant enzymes catalase, superoxide dismutase,
peroxidase and thiobarbituric acid reactive substances (TBARS) were
high in rat plasma and liver after feeding Haematococcus biomass as
source of astaxanthin. Astaxanthin in H. pluvialis offered the best
protection from free radicals in rats followed by β-carotene and lutein.
Astaxanthin contains a unique molecular structure in the presence of
hydroxyl and keto moieties on each ionone ring, which are responsible
for the high antioxidant properties.
Antioxidant activity of astaxanthin was 10 times more than
zeaxanthin, lutein, canthaxanthin, β-carotene and 100 times higher than
α-tocopherol. The oxo functional group in carotenoids has higher
antioxidant activity without pro-oxidative contribution. The polyene
chain in astaxanthin traps radicals in the cell membrane, while the
terminal ring of astaxanthin could scavenge radicals at the outer and
inner parts of cell membrane. Antioxidant enzyme activities were
evaluated in the serum after astaxanthin was supplemented in the diet of
rabbits, showing enhanced activity of superoxide dismutase and
thioredoxin reductase whereas paraoxonase was inhibited in the

11
oxidative-induced rabbits. Antioxidant enzyme levels were increased
when astaxanthin fed to ethanol-induced gastric ulcer rats.

2. Anti-Lipid Peroxidation Activity:

Astaxanthin has a unique molecular structure which enables it to
stay both in and outside the cell membrane. It gives better protection
than β-carotene and Vitamin C which can be positioned inside the lipid
bilayer. It serves as a safeguard against oxidative damage by various
mechanisms, like quenching of singlet oxygen; scavenging of radicals to
prevent chain reactions; preservation of membrane structure by
inhibiting lipid peroxidation; enhancement of immune system function
and regulation of gene expression. Astaxanthin and its esters showed
80% anti-lipid peroxidation activity in ethanol induced gastric ulcer rats
and skin cancer rats. Astaxanthin inhibited lipid peroxidation in
biological samples reported by various authors

3. Anti-Inflammation:
Astaxanthin is a potent antioxidant to terminate the induction of
inflammation in biological systems. Astaxanthin acts against
inflammation. Algal cell extracts of Haematococcus and Chlorococcum
significantly reduced bacterial load and gastric inflammation in H.
pylori-infected mice. Park et al. reported astaxanthin reduced the DNA
oxidative damage biomarker inflammation, thus enhancing immune
response in young healthy adult female human subjects.
Haines et al. reported lowered bronchoalveolar lavage fluid
inflammatory cell numbers, and enhanced cAMP, cGMP levels in lung
tissues after feeding astaxanthin with Ginkgo biloba extract and Vitamin

12
C. Another study showed astaxanthin esters and total carotenoids from
Haematococcus exerted a dose-dependent gastroprotective effect on
acute, gastric lesions in ethanol-induced gastric ulcers in rats. This may
be due to inhibition of H1, K1 ATPase, upregulation of mucin content
and an increase in antioxidant activities.
Astaxanthin showed protective effect on high glucose induced
oxidative stress, inflammation and apoptosis in proximal tubular
epithelial cells. Astaxanthin is a promising molecule for the treatment of
ocular inflammation in eyes as reported by the Japanese researchers.
Astaxanthin can prevent skin thickening and reduce collagen reduction
against UV induced skin damage .

4. Anti-Diabetic Activity:
Generally, oxidative stress levels are very high in diabetes
mellitus patients. It is induced by hyperglycemia, due to the dysfunction
of pancreatic β-cells and tissue damage in patients. Astaxanthin could
reduce the oxidative stress caused by hyperglycemia in pancreatic β-
cells and also improve glucose and serum insulin levels. Astaxanthin can
protect pancreatic β-cells against glucose toxicity. It was also shown to
be a good immunological agent in the recovery of lymphocyte
dysfunctions associated with diabetic rats.
In another study, ameliorate oxidative stress in streptozotocin-
diabetes rats were inhibited by the combination of astaxanthin with α-
tocopherol. It is also inhibited glycation and glycated protein induced
cytotoxicity in human umbilical vein endothelial cells by preventing
lipid/protein oxidation. Improved insulin sensitivity in both
spontaneously hypertensive corpulent rats and mice on high fat plus high

13
fructose diets was observed after feeding with astaxanthin. The urinary
albumin level in astaxanthin treated diabetic mice was significantly
lower than the control group. Some of the studies demonstrated that
astaxanthin prevents diabetic nephropathy by reduction of the oxidative
stress and renal cell damage.

5. Cardiovascular Disease Prevention:

Astaxanthin is a potent antioxidant with anti-inflammatory
activity and its effect examined in both experimental animals and human
subjects. Oxidative stress and inflammation are pathophysiological
features of atherosclerotic cardiovascular disease. Astaxanthin is a
potential therapeutic agent against atherosclerotic cardiovascular
disease.
The efficacy of disodium disuccinate astaxanthin (DDA) in
protecting mycocardium using mycocardial ischemia reperfusion model
in animals was evaluated. Myocardial infarct size was reduced in
Sprague Dawley rats, and improved in myocardial salvage in rabbits
after four days of pre-treatment with DDA at 25, 50 and 75 mg/kg body
weight. Astaxanthin was found in rat mycocardial tissues after
pretreatment with DDA at dosage of 150 and 500 mg/kg/day for seven
days. Astaxanthin effects on blood pressure in spontaneously
hypertensive rats (SHR), normotensive Wistar Kyoto rats (NWKR) and
stroke prone spontaneously hypertensive rats (SPSHR) were reported.
Astaxanthin was found in the plasma, heart, liver, platelets, and
increased basal arterial blood flow in mice fed with astaxanthin
derivative. Human umbilical vien endothelial cells and platelets treated
with the astaxanthin showed increased nitric oxide levels and decrease in

14
peroxynitrite levels. Mice fed 0.08% astaxanthin had higher heart
mitochondrial membrane potential and contractility index compared to
the control group. Astaxanthin effects on paraoxonase, thioredoxin
reductase activities, oxidative stress parameters and lipid profile in
hypercholesterolemic rabbits were evaluated. Astaxanthin prevented the
activities of those enzymes from hypercholesterolemia induced protein
oxidation at the dosages of 100 mg and 500 mg/100 g.

6. Anticancer Activity:
The specific antioxidant dose may be helpful for the early
detection of various degenerative disorders. Reactive oxygen species
such as superoxide, hydrogen peroxide and hydroxyl radical are
generated in normal aerobic metabolism. Singlet oxygen is generated by
photochemical events whereas peroxyl radicals are produced by lipid
peroxidation. These oxidants contribute to aging and degenerative
diseases such as cancer and atherosclerosis through oxidation of DNA,
proteins and lipids.
Antioxidant compounds decrease mutagenesis and carcinogenesis
by inhibiting oxidative damage to cells. Cell–cell communication
through gap junctions is lacking in human tumors and its restoration
tends to decrease tumor cell proliferation. Gap junctional
communication occurs due to an increase in the connexin-43 protein via
upregulation of the connexin-43 gene. Gap junctional communication
was improved in between the cells by natural carotenoids and retinoids.
Canthaxanthin and astaxanthin derivatives enhanced gap junctional
communication between mouse embryo fibroblasts.
Increased connexin-43 expression in murine fibroblast cells by β-

15
carotene was reported. Astaxanthin showed significant antitumor activity
when compared to other carotenoids like canthaxanthin and β-carotene.
It also inhibited the growth of fibrosarcoma, breast, and prostate cancer
cells and embryonic fibroblasts. Increased gap junctional intercellular
communication in primary human skin fibroblasts cells were observed
when treated with astaxanthin. Astaxanthin inhibited cell death, cell
proliferation and mammary tumors in chemically induced male/female
rats and mice. H. pluvialis extract inhibited the growth of human colon
cancer cells by arresting cell cycle progression and promoting apoptosis
reported by Palozza et al.
Nitroastaxanthin and 15-nitroastaxanthin are the products of
astaxanthin with peroxynitrite, 15-nitroastaxanthin anticancer properties
were evaluated in a mouse model. Epstein-Barr virus and carcinogenesis
in mouse skin papillomas were significantly inhibited by astaxanthin
treatment.

7. Immuno-Modulation:
Immune system cells are very sensitive to free radical damage.
The cell membrane contains poly unsaturated fatty acids (PUFA).
Antioxidants in particular astaxanthin offer protection against free
radical damage to preserve immune-system defenses. There are reports
on astaxanthin and its effect on immunity in animals under laboratory
conditions however clinical research is lacking in humans. Astaxanthin
showed higher immuno-modulating effects in mouse model when
compared to β-carotene.
Enhanced antibody production and decreased humoral immune
response in older animals after dietary supplementation of astaxanthin

16
was reported. Astaxanthin produced immunoglobulins in human cells in
a laboratory study. Eight week-supplementation of astaxanthin in
humans resulted in increased blood levels of astaxanthin and improved
activity of natural killer cells which targeted and destroyed cells infected
with viruses. In this study, T and B cells were increased, DNA damage
was low, and C-reactive protein (CRP) was significantly lower in the
astaxanthin supplemented group.

8. Commercial Applications of Astaxanthin:

In the present scenario, production of astaxanthin from natural
sources has become one of the most successful activities in
biotechnology. Astaxanthin has great demand in food,, nutraceutical and
pharmaceutical applications. This has promoted major efforts to improve
astaxanthin production from biological sources instead of synthetic ones.
According to the current literature, astaxanthin is used in various
commercial applications in the market. Astaxanthin products are
available in the form of capsule, soft gel, tablet, powder, biomass,
cream, energy drink, oil and extract in the market. Some of the
astaxanthin products were made with combination of other carotenoids,
multivitamins, herbal extracts and omega-3, 6 fatty acids.
Patent applications are available on astaxanthin for treatment of
bacterial infection, vascular failure, cardiovascular diseases, cancer,
inhibiting lipid peroxidation, inflammation, reducing cell damage and
body fat, and improving brain function and skin thickness. Astaxanthin
containing microorganisms or animals find many applications in a wide
range of commercial activities, the reason for which astaxanthin
enriched microalgae production can provide more attractive benefits.

17
 AIM AND OBJECTIVE

AIM:

The biological active compounds derived from natural

resourses that can be involved to control various diseases. So the aim of
the present study is isolation of astaxanthin from the shrimp shell waste.

OBJECTIVE:

1. To extract astaxanthin from shrimp shells by using organic

solvents,
2. To identifying it by performing thin layer chromatography and
3. To determine its phytochemical propertites and its anti microbial
activity on various micro organisms.

18
 MATERIALS AND METHODS

Collection of crustacean waste and raw material preparation

The crustacean waste was collected from shrimp processing
centre (costal cooperation limited), Visakhapatnam region of Andhra
Pradesh, and transported to laboratory under iced condition. Samples
were washed under running water and dried at 50°C in oven for 24
hours. They were packed in polyethylene bags and stored at - 20°C until
use. Dry samples were thawed in running water before use and
homogenized in a laboratory mixer prior to extraction of astaxanthin and
its estimation.

Figure no.5 Dried shrimp shell

19
Extraction of Shrimp Shell Waste by Using Different Solvents

Extraction of astaxanthin from crustacean shell waste was

investigated by using different organic solvents such as Methanol,
Petroleum ether, Chloroform, Hexane, and Acetone. 10 g of
homogenized dry crustacean shell waste were extracted with 100ml of
each solvent by stirring it for 1 hour on magnetic stirrer. Then the
carotenoid extract was filtered by using Whatman No.1 filter paper. The
recovered crustacean residues were again added with fresh solvent and
again filter it using whatman filter paper. This process was repeated until
the filtrate was colourless (i.e. a minimum of 3 times). Then the
collected extracts of each individual solvent were pooled together,
labeled and stored for further studies. The pooled extract of each organic
solvent is collected in separating funnel and 9.4ml of 0.73% (w/v) NaCl
solution were added to 100ml of each filterate. After through mixing the
epiphase was collected. Then to the lower phase an equal volume of
water was added, mixed and then the epiphase was collected. The
collected epiphase was pooled together and was kept in air for
evaporation these results in a semisolid coloured substance. This can be
further dried to get a complete powdered substance which can store for
further analysis.

20
 Figure no.6 Organic solvents with dissolved astaxanthin

Identification of Astaxanthin in Shrimp Shell Waste Extract by

Thin Layer Chromatography (TLC):
Identification of astaxanthin in the shrimp shell extract was done
using thin layer chromatography (TLC) based on the method of
Kobayashi and Sakamoto, 1999. For this, a small volume of the extract
was spotted on silica gel plate along with standard astaxanthin and Spot
development was done by using using acetone: hexane [3:7 (v/v)] as
mobile phase. The separated bands were identified by using standard
Astaxanthin (Source: Haematococcus pluvialis; Manufacturer: Zenith
nutrition, India) and internationally accepted Rf values for Astaxanthin
monoester and astaxanthin diester.

21
Quantification of Astaxanthin:
The organic solvent extracted astaxanthin was quantified by
measuring absorbance at 470 nm and using the equation of Kelley and
Harmon.
AST (µg /g) = A x D x 106 / 100 x G x d x E1cm1%
Where AST is astaxanthin concentration in µg/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);
E is extinction co-efficient, 2100.

Primary screening for phytochemical constituents:

The screening and study of six different sovent extracts for

phytochemical constituents was performed using generally accepted
laboratory technique for qualitative determinations. The constituents
screened for flavonoids, glycosides, steroids, tannins, phenols and
alkaloids. The distribution of these constituents in the crustacean extract
specimens were assessed and compared.

Test for Glycosides:

To 1ml of the extract add 2ml of acetic acid and then cooled in an
ice bath at 4°C. To this mixture 1ml of concentrated sulphuric acid was
added drop wise. The formation of an oil layer on top of solution
indicated the presence of glycosides (Odebiyi and sofowora et al.,
1978)

22
Test for alkaloids:

To 3ml of the extract add 1ml of 1% HCL. This resulting mixture

was then treated with few drops of Mayer’s reagent. The appearance of a
creamy white precipitate confirmed the presence of alkaloids (Ogukwe
et al., 2014)

Test for tannins:

Two drops of 5% ferric chloride was added to 1ml of the plant

extract. The appearance of a dirty-green precipitate indicated the
presence of tannins (Trease and Evans et al., 1996)

Test for Flavonoids:

To 1ml of the extact add 3 drops of ammonia solution followed by

0.5 ml of concentrated HCL. The resultant pale brown colouration of the
entire mixture indicated the presence of flavonoids (Odebiyi and
sofowora et al., 1978)

Test for steroids:

To 1ml of extract add 1ml of concentrated tetraoxosulphate acid.

A red colouration confirmed the presence of steroids (Trease and Evans
et al., 1996)

Test for phenols:

The extract (50mg) was dissolved in 5ml of distilled water. To

this, few drops of neutral 5% ferric chloride solution were added. A derk
green colour indicated the presence of phenolic compounds.

23
Test for Proteins:

a. Biuret test :

To 2 ml of filtrate is treated with 1 drop of 2 % copper sulphate

solution. To this 1 ml of ethanol 95 % is added, followed by excess of
potassium hydroxide pellets. Pink colour ethanolic layer indicates the
presence of protein.

Antimicrobial activity:
The antibacterial activity of the astaxanthin extracts was
determined in accordance with agar-well diffusion method. The bacterial
isolates were first grown in a nutrient broth for 24 hours before use.
Two hundred microliter of standardized cell suspensions were
spread on a nutrient agar plates. Wells were then bored into the agar
using a sterile 6mm diameter cork borer. Approximately 100 micro litres
of carotenoid extracts were introduced into the wells, allowed to stand at
room temperature for about 1 hour and then incubated at 37°C in an
incubator. Controls were setup in parallel using the solvents that were
used for extraction. The plates were observed for zone of inhibition after
24 hours. For my test purpose I have taken four different species of
micro organisms. They were:

01. Staphylococcus aureus

02. Klebsiella pneumonia

03. Escherichia coli

04. Streptococci

24
 Results and Discussion

Astaxanthin is an attractive compound in food, cosmetics, and

various pharmaceutical applications (Kim et al. 2011) since it has
several essential biological functions including strong potent antioxidant,
protection against UV light, enhance immune response, and strong
coloring agent (Guerin et al. 2003).

Astaxanthin is a xanthophylls carotenoid which is found in

various microorganisms and marine animals. In my study astaxanthin
extraction was done by using stranded method.

Extraction of Astaxanthin from crustacean shell wastes:

Extraction of astaxanthin from the crustacean shell wastes were

done with different organic solvents(Acetone,Methnol,and Hexane). The
solvent extracted carotenoid was in the form of an orange-red coloured
solution.

Figure no.7 Astaxanthin extracted through organic solvents

25
Identification of compounds by Thin layer chromatography:

Upon performing thin layer chromatography three bands were

obtained which belongs to three samples derived of acetone, methanol,
and hexane extracts, along with the fourth band which contains standard
astaxanthin that is dissolved in acetone. When compared with standard
astaxantin these three samples show similar Rf values. The Rf values for
three bands were 0.69, 0.67,0.67. These were clearly differentiated in the
Table 1. When compared these Rf values with internationally standard
values of astaxanthin these are nearly similar to Rf values of astaxanthin
Di-ester.

Figure no.8 Thin layer chromatography

26
Table no.2 Rf values of astaxanthin from different organic solvents
extracts:

S/no Solvent extract Rf value

1. Acetone 0.69
2. Methanol 0.67
3. Hexane 0.67
4. Standard 0.69

Quantification of Astaxanthin from crustacean shell wastes:

All three extracts of the crustacean shell extracts were shown

varying extraction yield of Astaxanthin by using equation of Kelley and
Harmon. The highest carotenoid yield of 64.7µg/g was obtained from
acetone extracts, when compared to methanol, hexane, whose yields
were 53.3µg/g and 44.2µg/g respectively shown in table no ... 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.

Table No:3 concentration of astaxanthin

S/no Extraction Solvent Astaxanthin in ug/g

1. Acetone 64.7 ug/g
2. Methanol 53.3 ug/g
3. Hexane 44.2 ug/g

27
 Fig 9:Astaxanthin in ug/g

70
60

Concentration
50
40
30 Astaxanthin in ug/g
20
10
0
Acetone Methanol Hexane
Extraction Solvents

Screening for Phytochemical constituents:

Investigations on the phytochemical screening of the
present different organic extract of crustacean shell waste were revealed
the presence of steroids and glycosides in all extracts. Carotenoids are
the compounds belongs to major group of terpenoids.
Fig10. Test for Steroids Fig 11. Test for glycosides

28
Fig 12 Test for proteins Fig 13 Test for phenols

Fig 14 Test for Tannins Fig 15 Test for flavonoids

29
Table No.4 PHYTOCHEMICAL SCREENING FOR CRUSTACEAN
SHELL EXTRACTS BY DIFFERENT ORGANIC SOLVENTS:

S/no Phytochemical compounds Acetone Methanol Hexane

1. Glycosides + + +

2. Alkaloids - - -

3. Tanins - - -

4. Flavonoides - - -

5. Steroids + + +

6. Phenols - - -

7. Proteins + + +

By performing phytochemical analysis it is observed that all the

three extracts(acetone, methanol, and hexane) had shown similar results.
Tests for Glycosides, proteins and steroids shown positive results and
the remaining tests were negative which indicates the absence of
alkaloids, tannins, flavonoides, and phenols.

Antibacterial activity:

Antimicrobial activity was studied by agar well diffusion method.

All three extracts were tested against both gram positive and gram
negative bacteria. The zone of inhibition were obtained from the all the

30
extracts were tabulated as follows. Acetone extracts had shown
maximum zone of inhibition followed methanol. The hexane extracts
does not show any visible zone of inhibition. To compare with the two
solvents, the highest zone of inhibition was present against klebsiella
pneumonia in both methanol and acetone etracts.

Acetone extracts:

The zone of inhibition ranged from 10 to 20mm in acetone extract

shown in the given table.

Table no.5 Acetone extracts of astaxanthin:

Test 50(µg/ml) 100(µg/ml) 150(µg/ml) 200(µg/ml)

Microorganisms (mm) (mm) (mm) (mm)
Staphylococcus 10 11 12 16
aureus
Streptococci 13 14 16 18
Klebsiella 15 15 18 20
pneumonia
Escherichia coli 12 13 15 16

Acetone extracts showed maximum zone of inhibition against

klebsiella pneumoniae (20mm) at 200µg/ml concentration followed by
streptococci (18mm), staphylococcus aureus (16mm) and Escherichia
coli(16mm) respesctively.

31
 Figure no 16. Anti microbial activity of Acetone extracts

Anti microbial activity of Acetone extracts

20
18
zone of inhibition in mm

16
14
12 Staphylococcus aureus
10
8 Streptococci
6 Klebsiella pneumoniae
4
2 Escherichia coli
0
50(µg/ml) 100(µg/ml) 150(µg/ml) 200(µg/ml)
(mm) (mm) (mm) (mm)
concentration of astaxanthin

32
Methanol extracts:

Table no.6 Methanol extracts astaxanthin:

Test 50(µg/ml) 100(µg/ml) 150(µg/ml) 200(µg/ml)

Microorganisms (mm) (mm) (mm) (mm)
Staphylococcus 10 10 14 16
aureus
Streptococci 10 12 13 16
Klebsiella 14 15 17 20
pneumonia
Escherichia coli 10 12 12 15

In the methanol extract the antimicrobial activity rangeged from 10 to

20mm indifferent concentrations such as 50 to 200µg/ml

Methanol extracts showed maximum zone of inhibition in

klebsiella pneumoniae (20mm) followed by staphylococcus aureus and
streptococci shown 16mm zone of inhibition, in Escherichia coli 15mm
zone of inhibition were observed.

Anti microbial activity of Methanol extracts

zone of inhibition in mm

20
18
16
14
12
10
8
6 50(µg/ml)
4
2
0 100(µg/ml) (mm)
150(µg/ml) (mm)
200(µg/ml) (mm)

Test organisms

33
 Figure no 16. Anti microbial activity of methanol extracts

Antimicrobial activity of astaxanthin was tested against four

strains of bacteria, two gram positive (S. aureus and B. subtilis) and two
gram negative (E.coli and E.aerogenes) in three different concentrations
(25 µg/ml, 50 µg/ml and 75 µg/ml) (Irna, C et al., 2017). A previous
study investigating the zone of inhibition of astaxanthin against E. coli
noted 12.06 mm as the finding, using acetone at 50 µg with a yield of
astaxanthin 29.01 µg/g. This was lower than the zone of inhibition of
astaxanthin from crab shells extracted using HPP at 50 µg/ml (Suganya
and Asheeba, 2015).

34
 In addition, others have reported the zone of inhibition of
astaxanthin against P. Aeruginosa, B. subtilis, and S. aureus, as being
22 mm, 18 mm, and 16 mm, respectively. These extracts were from
shrimp in Cochin, Kerala (India) using hexane, at 75 µg lower than the
zone of inhibition of astaxanthin extracted using HPP at 75 µg/ml
(Ushakumari and Ramanujan, 2013). All four strains of bacteria
produced zone of inhibition from the HPP method which was greater
compare to chemical extraction. High Pressure Processing was
susceptible to inhibit growth of both Gram-positive and Gram-negative
bacteria (Ushakumari and Ramanujan et al., 2013).
Ushakumari and Ramanujan 2013 study about the antibacterial
activity of astaxanthin from Shrimp of the species Metapenaeus
dobsoni. The antibacterial activity of the extract was studied by using
well diffusion method salmonella typhi produced 20 mm diameter for
zone of inhibition, pseudogonas aeroginasa 24 mm, bacillus subtilis 18
mm. staphylococcus aureus 16 mm. The extract showed excellent
antibacterial activity than the standard chloramphenicol. Among this
pseudomonas aeroginasa showed maximum inhibition.

35
 CONCLUSION
Shrimp shell wastes from industries can be utilized for the
isolation of important bioactive compounds like natural carotenoid such
as astaxanthin. From this above study acetone will be a good extraction
media for astaxanthin among organic solvents. TLC separation
confirmed that carotenoid extract from crustacean waste contained
astaxanthin diester. Potent antimicrobial activity of crustacean waste
extract may find application in pharmaceutical industries for treatment
or prevention of pathogenic microbial infections.

It is used as a nutritional supplement, antioxidant, anticancer and

anti microbial agent. Various commercial applications present in the
pharma industry using astaxanthin such as tablets, capsule, syrups, soft
gels, creams and granulated powder. It is also available in food, feed and
nutraceuticals applications. In the further research focused on effect of
astaanthin on various biological activities and their uses in nutraceuticals
and pharmaceutical applications. In future investigated on their
metabolic pathways and also molecular studies in invitro and invivo for
their use in commercial purpose.

36