American-Eurasian Journal of Scientific Research 8 (2): 63-67, 2013

ISSN 1818-6785
IDOSI Publications, 2013
DOI: 10.5829/idosi.aejsr.2013.8.2.7381
Corresponding Author: G. Sibi, R and D centre, Robust Materials Technology Pvt. Ltd., Bengaluru, Karnataka, India.

63
Preparation of Glucosamine Hydrochloride from Crustacean Shell
Waste and Its Quantitation by RP-HPLC
G. Sibi, K. Dhananjaya, K.R. Ravikumar, H. Mallesha, R.T. Venkatesha,
1 1 1 1 2
Dwijendra Trivedi, Khum Prasad Bhusal, Neeraj and Krishne Gowda
2 2 2 2
R and D Centre, Robust Materials Technology Pvt. Ltd. Bengaluru, Karnataka, India
1
Dayananda Sagar College of Biological Sciences, Bengaluru, Karnataka, India
2
Abstract: Various products derived from crustaceans have food and medicinal values. Glucosamine is an amino
monosaccharide acting as substrate for the production of aggrecan and proteoglycans and thus have
therapeutic activity in osteoarthritis. The present study has been aimed to prepare glucosamine hydrochloride
(Glu-HCl) from various crustacean shells namely Penaeus monodon (Indian shrimp), Portunus pelagicus (blue
crab) and Portunus sanguinolentus (three spot crab) by acid hydrolysis and its quantitation by reversed phase
high performance liquid chromatography (RP-HPLC). The yield of chitin after demineralization with 0.5M HCl
was 87.83%, 89.18% and 51.11% and deacetylation of chitin with 2N NaOH resulted in the yield of 68.91%,
75.67% and 30% for P.sanguinolentus, P.pelagicus and P.monodon respectively. HPLC analysis of obtained
glucosamine hydrochloride revealed species of Portunus (21.64 mg g and 21.83 mg g ) were better source
1 1
of Glu-HCl than P.monodon (3.32 mg g ). Further, this study describes the recycling of crustacean wastes to
1
a value added product which is having potential applications in the field of food and medicine.
Key words: Glucosamine HCl HPLC Crustaceans Chitosan Chitin
INTRODUCTION invertebrates and cell wall of fungi and is composed of
The major economically important group of Deacetylated form of chitin is known as chitosan which is
crustaceans includes lobsters, shrimps and crabs. About composed primarily of 2-amino-2-deoxy- -D-glucose
40-50% of total weight of crustaceans goes as waste while (glucosamine). Glucosamine is an amino monosaccharide
processing for human food and the slower degradation of acting as a preferred substrate for the constitution
crustacean shell waste has become the major concern in of glycosaminoglycan chains. It is also a substrate for
sea food processing industries [1, 2]. Further, it has the production of aggrecan and proteoglycans which
resulted in waste collection, disposal and pollution gives hydrophilicity to the cartilage thus beneficial in
problems [3]. Proper use of crustacean wastes allows treatment of osteoarthritis [16]. Glucosamine has anti-
recovery of value added by products which are having cancer [17, 18], anti-inflammatory [19] and antibacterial
potential applications in the field of food and medicine [20] effects.
[4-8]. Glucosamine has been prepared from various The present study was aimed to prepare glucosamine
crustaceans [9-10]. Glucosamine produced by hydrolysis hydrochloride using crustacean (Penaeus monodon,
of chitosan has therapeutic activity in osteoarthritis [11]. Portunus pelagicus and Portunus sanguinolentus) shell
Methods have been described for the quantitation of waste involving demineralization, deproteination and
glucosamine in chitin by HPLC [12-15]. deacetylation processes. Both quality and quantity of
Chitin is mainly produced from cuticles of various obtained Glu-HCl were determined by Fourier Transform
crustaceans mainly by crabs and shrimps. Chitin is the Infrared Spectroscopy (FT-IR) and Reversed Phase High
major structural component of exoskeleton of Performance Liquid Chromatography (RP-HPLC).
2-acetamido-2-deoxy- -D-glucose (N-acetyl glucosamine).
Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013
64
MATERIAL AND METHODS Agilent Technologies). The mobile phase used was
Demineralization: Crustacean (shrimp and crab) shells flow rate of 0.6 ml min in column at ambient temperature
were collected from local market and washed in cold water with infusion volume of 20 l in each experiment. Injection
tap water by removing dirt and loose tissues. Legs and volume was 10 l and detection was by UV absorbance at
heads were separated and the shells were dried in hot sun 195 nm.
for 24 hours. After drying, the shells were treated with Stock standard solutions were prepared by accurately
0.5M HCl (1:14 w/v) for 8 hours at room temperature and adding 100 mg of glucosamine hydrochloride to 50 ml
the squashy shells were rinsed in water to remove calcium mobile phase (orthophosphoric acid: acetonitrile - 70:30)
chloride. in a 100 ml volumetric flask, sonicated and made up to
Deproteination: The demineralized shells were treated phase, sonicated and filtered through 0.22m filter.
with 1N NaOH (1:12 w/v) at 90C for 2 hours and the
residue was neutralized in running tap water. The RESULTS AND DISCUSSION
deproteinized shells were dried in sun to obtain the chitin
material which was further used to produce chitosan by Glucosamine is part of the structure of chitosan and
deacetylation process. chitin which compose the exoskeletons of crustaceans,
Deacetylation: Chitin was treated with 2N NaOH in monomers of -(1-4)-linked-D-glucosamine (GlcN), an
(1:14 w/v) solution to remove the acetyl groups at room amino monosaccharide with physiological importance to
temperature for 8 hours in a shaker. The resulting chitosan the human body.
was washed in running tap water followed by distilled In the present study, crustacean wastes were used to
water and dried in sun. produce chitin and chitosan by acid hydrolysis method.
Preparation of Glucosamine HCL: The chitosan material 89.18% and 51.11% and deacetylation of chitin with 2N
was coarsely grinded and hydrolyzed with conc. HCl at NaOH resulted in the yield of 68.91%, 75.67% and 30% for
90C for 75 minutes. The resultant brownish black material P.sanguinolentus, P.pelagicus and P.monodon
was dissolved in distilled water and decolourized with respectively (Table 1).
activated charcoal. The solution was filtered and the The finger prints of the FT-IR spectra of standard and
filtrate was evaporated at 45C to recover glucosamine prepared Glu-HCl does not show any excess peaks (Fig 1).
hydrochloride (Glu-HCl). The crystals were washed in The absorption bands at particular wavelength are due to
ethanol and dried at 50C in hot air oven and analyzed by the formation of NH in Glu-HCl which was in accordance
high performance liquid chromatography. with previous data [21].
FT-IR Spectrum: Standard glucosamine hydrochloride comparing with the retention time of pure standard. Purity
and obtained Glu-HCl were compared using FT-IR of the peaks was confirmed with the characteristic spectra
spectra for identification and comparing purity. obtained from the detector. Fig 2 represents the
The FT-IR spectrum was recorded on a Cary 640 FT-IR chromatograms glucosamine hydrochloride prepared from
(Agilent Technologies) in the form of Kbr discs. crustacean shells. In the figures, y axis is milli absorbance
The resolution was in 2 cm and the scanning range was unit (mA) and x axis is retention time (RT) in minutes.
1
4000-800 cm . There were two peaks occurred close which are related to
1
HPLC Method: HPLC analysis was performed (Fig 2). From the HPLC analysis, the yield of Glu-HCl
using 1260 infinity series LC system installed with prepared from crustacean wastes was 21.64 mg g , 21.83
a G1311C pump, a G1329B autosampler and a G13166 mg g and 3.32 mg g .
column compartment and G4212B DAD Detector. Previous studies have revealed that acid hydrolysis
Chromatographic separation was carried out on a Zorbax is the preferred method to release glucosamine from chitin
Eclipse XDB-C8 Analytical column (4.6 250mm, 5 ; material [22]. Several factors such as time, temperature,
orthophosphoric acid (pH 2.5): acetonitrile (70:30) with the
1
final volume. Working standards were prepared in mobile
arthropods and fungi. The hydrolysis of chitosan results
The yield of chitin after demineralization was 87.83%,
3
+
Peak identification of glucosamine was done by
adsorption of glucosamine in the standard chromatogram
1
1 1
Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013
65
Fig. 1: FT-IR spectra of Glu-HCl
Fig. 2: HPLC chromatograms of glucosamine hydrochloride from crustacean waste
Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013
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Table1: Yield of chitin, chitosan and Glu-HCl from crustacean waste
Sample Chitin Chitosan Glu-HCl
Portunus sanguinolentus 87.83% 68.91% 21.64 mg g
1
Portunus pelagicus 89.18% 75.67% 21.83 mg g
1
Penaeus monodon 51.11% 30% 3.32 mg g
1
pH and acid concentration are attributed to the release of
glucosamine from chitin or chitosan. Acid hydrolysis of
chitin with concentrated HCl for longer time leads to
breakdown of glucosamine and decreased recovery [23].
Low concentrations of hydrochloric acid slow down the
chitin hydrolysis. Mojarrad et al. [15] described the
optimized conditions for the preparation of glucosamine
HCl as 30% and 37% HCL (9:1 v/w) for 4 hours from
Metapenaeus monoceros. In the present study, HCl
(0.5M) treatment for 8 hours produced a better yield of
chitin (89.83 %) from P.pelagicus. Purchase and Braun
[24] isolated glucosamine from crab shells. Stacey and
Webber [25] described the simple production of
glucosamine from crab shells of low protein content.
The present findings revealed that exoskeleton of
P.sanguinolentus and P. pelagicus were better sources
of Glu-HCl than P.monodon under the described
conditions. Ferrer et al. [26] has obtained 80% yield of
glucosamine during the acid hydrolysis of shrimp shell
wastes. The yield of chitin, chitosan and Glu-Hcl from
P.monodon was comparatively lower than that of crab
species used in this study.
CONCLUSION
The present study was found as simple, efficient and
suitable for the preparation of glucosamine from
crustacean shells thereby recycling crustacean wastes.
Further, HPLC method allows the detection and
quantitation of glucosamine prepared by acid hydrolysis
of chitosan from exoskeleton of crustaceans and could be
used as routine method for analysis of Glu-HCl in raw
materials and pharmaceutical formulations.
ACKNOWLEDGMENT
The authors are grateful to Vision Group on Science
and Technology (VGST), Government of Karnataka, India
for the financial support under SPiCE scheme.
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