Cuttlebone: Characterization

and Applications

By: Safieh Momeni

 Cuttlebone
Cuttlebone signifies a special class of ultra-lightweight, high stiffness and high
permeability cellular biomaterials, providing the cuttlefish with an efficient means of
maintaining neutral buoyancy at considerable habitation depths. In addition, this rigid
cellular material provides the structural backbone of the body and plays a key role in the
protection of vital organs.
Cuttlebone

The cuttlebone has two main components:

 Dorsal shield
 Lamellar matrix

 The dorsal shield is very tough and dense, providing a rigid substrate for protection,
structure and the development of the lamellar matrix of cuttlebone.

 The lamellar matrix of cuttlebone has an extreme porosity (up to 90%), but also
manages to withstand very high hydrostatic pressure.
Lamellar matrix
 The lamellar matrix consists primarily of aragonite (a crystallised form of
calcium carbonate, CaCO3), enveloped in a layer of organic material composed
primary of β-chitin.
 The organic layer entirely envelopes the inorganic ceramic, and is thought to
initiate, organise and inhibit the mineralisation of the inorganic material. From
a mechanical perspective, the organic layer is also thought to provide a certain
toughening effect to the material
Applications

Due to the unique physical, chemical and mechanical characteristics

of this natural cellular material, a range of novel applications for
the material have recently been investigated.
Preparation of highly porous hydroxyapatite from
cuttlefish bone

 Hydroxyapatite structures for tissue engineering applications have been produced by

hydrothermal (HT) treatment of aragonite in the form of cuttlefish bone at 200 ̊C.
Aragonite (CaCO3) monoliths were completely transformed into hydroxyapatite after
48 h of HT treatment.

 The substitution of CO32- groups predominantly into the PO43- sites of the
Ca10(PO4)6(OH)2 structure was suggested by FT-IR spectroscopy and Rietveld structure
refinement. The SEM micrographs have shown that the interconnected hollow structure
with pillars connecting parallel lamellae in cuttlefish bone is maintained after
conversion. Specific surface area (SBET) increased and mean pore size decreased by
HT treatment.

J Mater Sci: Mater Med (2009) 20:1039–1046

Hydroxyapatite
 Hydroxyapatite (HAP; Ca10(PO4)6(OH)2) due to its similarity with the mineral phase of
bone has been studied for many years as an implant material . In recent years, particular
attention has been paid to the synthesis of HAP ceramics with the porous morphology
required for vascularization, bone cell invasion, and angiogenesis, which further
improve its biomedical properties.
 The HAP structure can incorporate a wide variety of ions implying a variable defect
structure of HAP. In biological apatites, or apatites originating from biogenetic
materials, the presence of other mineral ions, such as Na+,K+,Mg2+, F- as well as
substitution of carbonate ions into the HAP structure is very common.
 The carbonate ion substitutes either at the phosphate tetrahedron (B-type substitution)
or at the hydroxyl site (A-type substitution) . The biological apatite that constitutes
bone mineral is considered to be of mixed AB-type substitution .
Hydroxyapatite
 While synthetic materials have been widely used in the biomedical field with great
success, natural structural materials are now providing an abundant source of novel
biomedical applications.
 During the last decade, an increased understanding of biomineralization has initiated
improvements in biomimetic synthesis methods and production of new generation of
biomaterials. The use of natural biogenic structures and materials such as corals,
seashells, animal bones, etc., for medical purposes has been motivated by limitations in
generating synthetic materials with the requisite structure and mechanical integrity.
Hydroxyapatite

 Dry cuttlefish bone was cut into small pieces about 2 cm3 large and was first heated to
350 ºC for 3 h to remove the organic component, and then sealed with the 15 ml
aqueous solution of 0.6 M NH4H2PO4 in Teflon lined stainless steel pressure vessel at
200ºC for various times (1–48 h).

 The stoichiometrically required aqueous solution of NH4H2PO4 to set the ratio Ca/P =
1.67 was determined using the data of DSC and TG analysis of raw cuttlefish bones.
The formed HAP was washed with boiling water and dried at 110ºC.
SEM micrographs of: a cuttlefish bone heated at
350C for 3 h. The corrugated appearance of the
pillars is shown in the inset, b the cuttlefish bone
after hydrothermal conversion at 200 C/24 h
showing plate- and needle-like HAP crystals (inset)
 Biotemplated Syntheses of Macroporous
Materials for Bone Tissue Engineering Scaffolds
and Experiments in Vitro and Vivo

ACS Appl. Mater. Interfaces

 The macroporous materials were prepared from the transformation of cuttlebone as
biotemplates under hydrothermal reactions. Cell experimental results showed that the
prepared materials as bone tissue engineering scaffolds or fillers had fine
biocompatibility suitable for adhesion and proliferation of the hMSCs (human marrow
mesenchymal stem cells).

 Histological analyses were carried out by implanting the scaffolds into a rabbit femur,
where the bioresorption, degradation, and biological activity of the scaffolds were
observed in the animal body.

 The prepared scaffolds kept the original three-dimensional frameworks with the ordered
porous structures, which made for blood circulation, nutrition supply, and the cells
implantation. The biotemplated syntheses could provide a new effective approach to
prepare the bone tissue engineering scaffold materials.
 Design and synthesis of new porous scaffold materials for bone rehabilitation are of
great significance since large quantities of skeletal reconstructive surgical cases need to
be performed with the scaffold materials each year worldwide.

 In bone healing occurrence, the graft materials play the crucial roles. The autologous
bones, as the gold-standard of the graft materials, can provide the scaffolds and active
factors for bone ingrowth.

 However, aside from the source of the autologous bone being greatly limited, the
autologous bone grafts are associated with an 8−39% risk of complications, e.g.,
hematoma, additional injury, superinfection, surgical complication, postoperative pain,
and chronic pain at the donor sites.

 Therefore, autologous bone grafts are normally not recommended for elderly or
pediatric patients or for patients with malignant or infectious disease.
 Alternative strategies, like allo- or xeno-transplantations, have major biocompatibility
disadvantages compared with autografts.
 To overcome these limitations, the bone graft substitutes have been used to reconstruct
bone defects. The perfect bone substitutes are osteoinductive, osteoconductive,
biocompatible, and bioresorbable, which should induce minimal or no grafts rejection
and can undergo remodeling and support new bone formation.
 On the other hand, it should be cost-effective and available in the amount required. The
bone substitutes as scaffolds are in favor of the bone cells migration, proliferation, and
new bone formation.
 Calcium phosphate-based ceramics are currently available and widely used in trauma
and orthopedic surgery for bone substitutes due to their chemical similarity to bone
mineral with minimal immunologic reactions, no foreign body reactions, or no systemic
toxicity. Hydroxyapatite (HA, Ca5(PO4)3(OH)) and beta tricalciumphosphate (β-TCP,
Ca3(PO4)2) are well-known bioceramics that possess high tissue compatibility and
osteoconductivity.
 However, HA seems to be too stable in vivo because it shows a similar crystalline phase
as bone mineral, which would be hard to tend toward chemical and biological
equilibrium with bone tissue.
Preparation of the Scaffold Materials

Cuttlebones (CB, 100 g, 30 × 10 × 3.5 cm) from the sampan-like spine of cuttlefishes were
cut into different dimensional cylinders. The mixture of pieces of dry cuttlebone and
(NH4)2HPO4 (mol/mol, 1:1.1) in aqueous solution were sealed in a 100 mL stainless
steel autoclave with Teflon liner and placed in a temperature-controlled electric furnace
at 180 °C (heating and cooling rates were 10 K·min−1). Different times of hydrothermal
reaction were tested between 3 and 48 h in independent experiments. The pH of the
solution was 7.8−8.2 before being placed in autoclaves and slightly lower after the
hydrothermal reaction.
 FTIR spectra of treated cuttlebone (a) and the scaffold
transformed from hydrothermal reaction at 180 °C for 3 h
(b), 6 h (c), 12 h (d), 24 h (e), and 48 h (f).

XRD patterns. Aragonite (ICDD card 01-071-2396)

(a), cuttlebone (b), hydroxyapatite (ICDD card 00-
009-0432) (c), and hydrothermal reaction for 3 h (d),
6 h (e), 12 h (f), and 24 h (g).
TG-DTA curves. (a) Cuttlebones; (b) scaffold materials transformed from cuttlebones
by hydrothermal reactions.
(a) Scanning microscope view of the sintered scaffold transformed from the cuttlebone
by hydrothermal reaction, and all the trabeculares were broken in the holes; (b) original
magnification of 400× clearly reveals the ordered macroporous framework supported by
the S-shaped pillars; (c) the floor supported by the “S” oriented walls; (d) transverse
section view of the magnification of 10 000× of the floor with 2−5 μm openings; (e)
image of exterior surface of the floor wall; (f) view of the magnification of 30 000× for
the ceramic floor.
(a) Top view of the scaffold; (b) the top view of the “S” oriented wall; (c) the nanocrystallite
composites at the top fracture surface of the “S” oriented walls.
In Vitro Anticoagulation Properties
SEM images of the proliferation and adhesion on the scaffold surfaces: (a) cell
inoculation for 2 d and (b) 6 d.
Histological view of the specimens in vivo. (a) Two weeks; (b) four weeks. Sections
were stained with eosin. Asterisks (∗) represented the implanted scaffolds. Black dots
represented the bone cells.
PCL-coated hydroxyapatite scaffold derived from cuttlefish bone:
Morphology, mechanical properties and bioactivity

In the present study, poly(ε-caprolactone)-coated hydroxyapatite scaffold derived from cuttlefish

bone was prepared. Hydrothermal transformation of aragonitic cuttlefish bone into hydroxyapatite
(HAp) was performed at 200 °C retaining the cuttlebone architecture. The HAp scaffold was coated
with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique.

Bioactivity was tested by immersion in Hank's balanced salt solution (HBSS) and mechanical tests
were performed at compression. The results showed that PCL-coated HAp (HAp/PCL) scaffold
resulted in a material with improved mechanical properties that keep the original interconnected
porous structure indispensable for tissue growth and vascularization. The prepared bioactive
scaffold with enhanced mechanical properties is a good candidate for bone tissue engineering
applications

Materials Science and Engineering C 34 (2014) 437–445

 Synthetic calcium phosphates, in particular hydroxyapatite , are the most commonly used
ceramics in dentistry and bone repair due to their chemical similarity to the inorganic matrix
of natural bone, excellent osteoconductivity and bioactivity. The production of porous HAp
scaffolds is still a theme of high relevance in the field of biomaterials science and technology.
Currently, porous HAp scaffolds have been prepared by a number of manufacturing
techniques including polymer foam replication, sol–gel and freeze casting, solid free-form
fabrication, etc. However, these methods are expensive and not well defined concerning the
internal porous architecture.
 The major drawback of the HAp scaffolds is their poor mechanical properties, especially the
brittleness and low fracture toughness. Therefore, they cannot be used in load bearing
application. To overcome these disadvantages HAp has been combined with polymers that
provide flexibility to the brittle system.
 A wide range of enzymatically and hydrolytically degradable polymers have been proposed
for biomedical applications, either natural (gelatine, chitosan, alginate) or synthetic (poly(ε-
caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA),etc. Synthetic
polymers are preferable to natural-based polymers for their tailorable designs, reproducible
degradation characteristics and wide range of mechanical properties.
 Impregnation/coating of scaffold with polymer is a very simple and economical method
that can improve the mechanical properties of porous HAp scaffold while preserving
the connectivity of the pores which is crucial for its application in BTE.

 Due to its biodegradability, biocompatibility, appropriate mechanical properties, and

low emission of harmful byproducts PCL has been widely used in the biomedical field
for the manufacture of tissue engineering supports .
Preparation of HAp/PCL composite scaffolds
 Thermal decomposition of HAp and HAp/PCL scaffolds

Weight loss of the HAp/PCL composite scaffold until 550 °C can be ascribed to the three-step
degradation process of PCL which decomposes to carbon dioxide, water, carbon monoxide and
short-chain carboxylic acids. The results showed that the composite HAp/PCL scaffold
contains 48.7±0.1wt.% of PCL.
SEM micrograph of raw cuttlefish bone (a, b) and
cuttlefish bone after HT conversion into HAp (c–f). (a)
Detail of lamellar matrix transverse cross-section (b)
channels formed by convoluted pillar (c, d) transverse
cross-section clearly displaying interconnected channeled
structure maintained after HT conversion, (e) roughly
spherical aggregates of HAp crystals and (f) dandelion-like
structures.
SEM micrographs of PCL coated HAp scaffold. After
polymer impregnation the interconnectivity of the
channels in HAp scaffold is maintained, (a)–(c). A PCL
layer on the Hap aggregates that resemble the
cauliflower morphology is evident, (d) and (e).
 The composite scaffold was exposed to Hank's balanced salt solution (HBSS),
in order to investigate its ability to induce the precipitation of biologically
active bonelike calcium phosphate layer on its surface.

 After 28 days of immersion in HBSS solution both HAp and HAp/PCL

scaffolds exhibited mineral formation.
SEM micrographs of the scaffolds' surface after
soaking in HBSS for 28 days: Mineralization on the
HAp (a, b) and HAp/PCL substrates (c–f).
 EDX analysis of the precipitated particles on the HAp surface revealed the presence of
P, Ca, O and minor amounts of Na+,Mg2+,K+ and Cl− ions, similar to the composition of
natural apatite from human bone. The Ca/P molar ratio values obtained from eight EDX
analyses range from 1.3 to 1.8 indicating the presence of calcium phosphate (CP)
phases that produce the bonelike apatite.

 Regarding the calcium phosphate mineralization on the HAp/PCL surface it is believed

that nucleation occurs heterogeneously by complexation of the calcium ions and the
negatively charged carboxylate groups formed during hydrolysis of the ester groups in
the polymer.
Wound Healing Effect of Cuttlebone Extract in Burn Injury of Rat

 Burn injury, one of the most common diseases in primary care, is also a major cause of
death and disability. The aim of this study was to evaluate the effect of cuttlebone (CB)
extract in thermal burn wounds in rats and to compare its effects with those of silver
sulfadiazine (SSD), the most widely used burn treatment.
 Burn injury was produced in rats by immersion of the shaved dorsal area to hot water.
CB and SSD significantly increased re-epithelialization in burn wounds and decreased
WBC levels after 14 days of treatment.
 By FT-IR, we characterized chitin the main component of CB. Taken together, these
results suggest the wound healing effects of CB and its therapeutic value in the
treatment of burn injury.

Food Sci. Biotechnol. 22(S): 99-105 (2013)

Wound Healing Effect of Cuttlebone Extract in Burn Injury of Rat

Wound healing effect of CB on burn injury in rats.

After burning with hot water, animals were treated
with CB by dermal application and the surface area
of wounds were observed at indicated times. (a)
negative control: no treatment after burn injury, (b)
white petroleum, (c) SSD, and (d) CB

Food Sci. Biotechnol. 22(S): 99-105 (2013)

Histological changes of the injured skin after
burn. Wound areas including a part of normal
tissue on each time points after burn injury were
extracted, fixed, and then stained by
Hematoxylin-Eosin. Samples were observed by
using a microscope (original magnification ×200).
(a) negative control: no treatment after burned
injury, (b) white petroleum, (c) SSD, and (d) CB.
Bar=40 μm
FT-IR spectrum for analysis of cuttlebone
 Chitin from the Extract of Cuttlebone Induces Acute Inflammation and
Enhances MMP1 Expression
 The extract from cuttlebone (CB) has wound healing effect in burned lesion of rat. In present study, the
main component of CB extract was analyzed and its wound healing activity was evaluated by using in
vitro acute inflammation model.

 The extract of CB stimulated macrophages to increase the production of TNF-α. The extract also
enhanced the production of TGF-β and VEGF, which were involved in angiogenesis and fibroblast
activation. The treatment with CB extract enhanced proliferation of murine fibroblast.

 CB extract also induced the activation of fibroblast to increase the secretion of matrix metalloproteases 1
(MMP1). The constituent of CB extract which has wound healing activity was identified as chitin by
HPLC analysis. The mechanism that the CB extract helps to promote healing of burned lesion is
associated with that chitin in CB extracts stimulated wound skins to induce acute inflammation and to
promoted cell proliferation and MMP expression in fibroblast. Our results suggest that CB or chitin can
be a new candidate material for the treatment of skin wound such as ulcer and burn.

Biomol Ther 21(3), 246-250 (2013)

The cytotoxicity of CB extract in (A) RAW 264.7
cell and (B) fibroblast. The cells were treated with
the extract at a dose dependent manner for 24 h, and
media containing MTT were added for measurement
of cell viability. Data are representative of at least
three independent experiments, each done in
triplicate. *p<0.05, **p<0.01
compared to non-treated cells.
 The mechanisms underlying wound healing processes involve the acute inflammatory
mediators. The new formation of blood vessels (angiogenesis) is necessary and a vital
component in wound healing. The migrating fibroblasts fill the wounded site and
stimulate the formation of granulation tissue. TNF-α has a pivotal role in the activation
of vascular endothelial cell, the induction of angiogenesis, and proliferation of
fibroblast.
 TGF-β and VEGF are involved in fibroblast migration and angiogenesis, respectively.
Therefore we first examined the in vitro mechanism of CB extract on wound healing
and accessed whether the wound healing effect of CB extract is due to the activation of
macrophages to produce cytokines, TGF-β and VEGF.
 RAW 264.7 cells were treated with CB extract and the levels of TNF-α, TGF-β and
VEGF were measured with the cell culture supernatant. The treatment with CB extract
induced the activation of macrophages and increased the production of TNF-α, TGF-β
and VEGF in macrophages at dose dependent manner.
The effect of CB extract on the production of cytokines
by macrophages. RAW264.7 cells were treated with the
CB extract at a dose dependent manner for 24 h, and cell
culture supernatants were collected for (A) TNF-α, (B)
TGF-β, and (C) VEGF assay. Data are representative of
at least three independent experiments, each done in
triplicate. *p<0.05, **p<0.01 compared to non-treated
cells.
Effect of CB on fibroblast proliferation. The
cells were treated with the extract at a dose
dependent manner for indicated times, and cell
numbers were measured using (A) the trypan
blue exclusion method and (B) microscopic
observation. Data are representative of at least
three independent experiments, each done
in triplicate.
 Chitin activates macrophage by interacting with cell surface receptors such as mannose
receptor and toll-like receptor-2. Chitin-activated macrophage enhances the formation
of tissue in the wound and the production of endothelial growth factor, consistent with
our results. Moreover, chitin is known to play an essential role in hemostasis.
Cuttlebone as reinforcing filler for natural rubber

 Cuttlebone was proved to be a biomass for new reinforcing filler for natural rubber (NR).
The cuttlebone particles were obtained by crushing cuttlebone and followed by sieving.
Density and crystal structure of the cuttlebone were 2.70 g/cm3 and an aragonite form of
CaCO3, respectively.

 The cuttlebone particles did not prevent a peroxide cross-linking reaction of NR, and
mechanical properties of peroxide cross-linked NR filled with cuttlebone particles were
found to be comparable with those of peroxide cross-linked NR filled with commercial
CaCO3 filler.

 Presence of chitin on the surface of the cuttlebone particles was speculated to result in a
good interaction between cuttlebone particles and NR, which may be ascribed to the
mechanical properties of cuttlebone filled NR samples.

European Polymer Journal 44 (2008) 4157–4164

 Natural rubber (NR) is one of the important elastomers and widely utilized to prepare
many rubber products. NR is often reinforced by incorporation of filler to improve its
mechanical properties: modulus, hardness, tensile strength, abrasion resistance and tear
resistance, and so on.

 Reinforcing fillers most often used are carbon black and silica (SiO2). Calcium
carbonate (CaCO3) is also utilized as filler for rubber.

 Efficiency of the reinforcing filler depends on several factors such as particle size,
surface area and shape of filler.
 In Thailand, fishermen harvest cuttlefish for food. Skeleton of cuttlefish is removed
during cooking, which results in large amounts of waste products of cuttlebone. Can we
use the cuttlebone as a new biomass? Since cuttlebone is mainly composed of CaCO3
and chitin, cuttlebone may be used like CaCO3 and chitin.
XRD patterns of (a) cuttlebone particles and (b) commercial CaCO3
filler (Silver W).
 Generally, the smaller the size of filler is, the larger the reinforcement effect of the filler
becomes. In this study, however, the average size of cuttlebone particles was about six
times larger than that of Silver W, but the reinforcement effect is comparable with that
of SilverW.

 Probably, the presence of organic component such as chitin is speculated to give a good
reinforcement effect of cuttlebone particles to NR. The interaction between the organic
part of cuttlebone and NR must be large, which is supported by SEM observation.
SEM images of peroxide cross-linked NR filled with
cuttlebone particles and commercial CaCO3 filler (Silver
W).
A cuttlebone-derived matrix substrate for hydrogen
peroxide/glucose detection

Classical steps involved in the fabrication of CDMSs: (a) digital photograph of a dorsal-view cuttlebone cut though the cross-section and (b)
schematically transverse section through the cuttlebone; (c) digital photograph of a block cut from lamellar part of the bone; (d) SEM image
showing the chamber-like architecture of the area marked in (c); (e) digital photograph of cuttlebone-derived matrix after decalcification (two
red arrows indicate the detached lamella, respectively); (f) digital photograph of several detached substrates presented in anaquarium dish.

Biosensors and Bioelectronics 25 (2009) 362–367

Schematic illustration of the fabrication of gold-functionalized CDMS and its optical biosensing principle based on the
biocatalytic GNPs growth in the presence of glucose. (1) Transparent CDMS, (2) functionalization of CDMS with gold seeds,
(3) H2O2-mediated enlargement of GNPs on sensor for detection.
(a) Absorbance spectra of the gold-functionalized CDMSs upon reaction with 1×10−4 M HAuCl4 in phosphate buffer (pH
7.4, 0.02M) that includes H2O2 of different concentrations at room temperature: (1) 0M, (2) 2×10−6 M, (3) 5×10−6 M, (4)
1×10−5 M, (5) 2.5×10−5 M, (6) 5×10−5 M, (7) 7.5×10−5 M, (8) 1×10−4 M, (9) 1.5×10−4 M. (b) A digital photograph
showing the differences in color of resulting modified substrates corresponding to the absorbance spectra 1–9, from the
bottom up. (c) Calibration curve corresponding to the absorbance at = 530nm of the gold-functionalized CDMSs upon
analyzing H2O2 of variable concentrations. Error bars are the standards of three replicates.
(a) Absorbance spectra of the gold-functionalized CDMSs upon reaction with 1×10−4 M HAuCl4, 12gmL−1 GOx in 0.02M
phosphate buffer that includes -d(+) glucose of different concentrations: (1) 0M, (2) 5×10−6 M, (3) 1.3×10−5 M, (4) 2.5×10−5 M,
(5) 3.5×10−5 M, (6) 5×10−5 M. (b) Digital image showing the visible differences in color of substrates corresponding to the
spectra (indicated by 1, 2, 3, 4, 5, 6). For all experiments the reaction time interval was 20min, 32±1 ◦C. (c) Calibration curve
corresponding to the absorbance at = 530nm of the gold-functionalized CDMSs upon analyzing -d (+) glucose of variable
concentration. Error bars are the standards of three replicates.
Direct formation of silver nanoparticles in cuttlebone-
derived organic matrix for catalytic applications
 Development of biologically derived materials for the construction of materials with new
functions is a crucial intersection of materials science and biotechnology, which is currently a
topic of research interest.
 In this paper, they report on the use of cuttlebone-derived organic matrix (CDOM) as scaffold
and reducer for the formation of silver nanoparticles (AgNPs). The experiment was carried out
by simple immersing of CDOM in tollen’s reagent and incubating at 80 ◦C.
 UV–vis spectra and TEM were utilized to characterize the AgNPs and investigate their
formation process. Results demonstrate that the size and distribution of AgNPs are influenced
by the incubation time and protein component in CDOM.
 Furthermore, the AgNPs–CDOM composite was applied to catalyze the reduction of 4-
nitrophenol in the presence of NaBH4, and it can be easily separated from the liquid-phase
reaction system during the reusing cycles.

Colloids and Surfaces A: Physicochem. Eng. Aspects 330 (2008) 234–240

(A) The whole appearance of cuttlebone after incision; (B) the schematic representation of cuttlebone’s transverse
section; (C) SEM image of cuttlebone’s framework; (D) digital pictures of the cuttlebone-derived organic matrix
(CDOM); (E) microscope picture of the organic matrix; (F) SEM image of the freeze-dried CDOM.
Evolution of the UV–vis spectra and digital images during the formation of silver nanoparticles in CDOM.
TEM images (A–C) and size distribution (D) of
AgNPs in CDOM1. Synthesis condition: 3mM
tollen’s reagent (Ag(NH3)2NO3), reacted for 1 h, 2
h and 4 h respectively. (A) 1 h (dark field image);
(B) 2 h; (C and D) 4 h.
Successive UV–vis absorption spectra of the reduction of 4-nitrophenol by NaBH4 using AgNPs–CDOM composite as
catalyst.
Plot of the conversion rate as a function of time during
reaction within different reuse cycles.
Physicochemical Characterization and Antioxidant Efficacy of
Chitosan from the Internal Shell of Spineshell Cuttlefish Sepiella
inermis
 Cuttlefish chitosan was extracted from the cuttlebone of Sepiella inermis by demineralization and deproteinization
and produced by deacetylation, and its physical and chemical parameters were also compared with that of
commercial chitosan. Ash, moisture, and mineral and metal content of the chitosan was estimated by adopting
standard methodologies. The rate of deacetylation was calculated as 79.64% by potentiometric titration. Through
viscometry and gel permeation chromatography, the molecular weight of chitosan was found to be significantly
lower than that of the commercial chitosan. Optical activity was found to be levorotatory. The structure of the
chitosan was elucidated with spectral techniques such as Fourier-transform infrared (FT-IR) and nuclear magnetic
resonance (NMR) spectroscopy. Cuttlefish chitosan showed a melting endothermic peak at 117.32C. The x-ray
diffraction (XRD) pattern of chitosan and standard chitosan exhibited the same crystalline peaks. Through scanning
electron microscopy (SEM) the fine structure of chitosan was studied. The binding capacity (water and fat) of
cuttlefish chitosan was found to be significantly higher than that of the commercial chitosan. The antioxidant
efficacy of chitosan was determined through the conjugated diene method, scavenging ability on DPPH radicals,
reducing power, and chelating ability on ferrous ions. This study has brought out the importance of shell as a
potential source for obtaining another natural antioxidant.

Preparative Biochemistry & Biotechnology, 43:696–716, 2013

Thanks