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International Journal of Marine Science 2013, Vol.3, No.37, 295-305
http://ijms.sophiapublisher.com
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1.5 Hydroxyproline assay and collagen quantification
Different forms of collagens
viz
., salt soluble (SS),
acid soluble (AS) and insoluble (Ins) collagens were
extracted by the modified method of Prockop (1964).
The content of hydroxyproline (Hyp) and total
collagen was quantified according to the slightly
modified methods as described earlier (Pallela et al.,
2011; Jamall et al., 1981; Siddiqi and Alhomida, 2003).
Briefly, the sponge tissues were cut into small pieces
and homogenized in sufficient distilled water to yield
10% homogenate (w/v). Equal volume of 12 N HCl
was added to each aliquot (2 mL) of homogenate and
hydrolyzed at 105
℃
for 18 h. Aliquots of 25 µL
sponge tissue hydrolyzate, in triplicate, were added to
separate vials and subjected to evaporation leading to
dryness under vacuum. Sets of samples were spiked
with 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.6 µg of
standard hydroxyproline (Hyp). To each of the
hydrolyzate and Hyp standard vials, 1.2 mL of 50%
isopropanol was added, followed by 0.2 mL of 0.56%
buffered chloramine-T solution. One ml of Ehrlich’s
Reagent (ER) solution was then added after an interval
of 10 mins. The solutions obtained were mixed and
incubated at 50
℃
for 90 mins. Samples were cooled
and the absorbance was measured spectrometrically at
558 nm, using water as reference and corrected for
reagent blank. The absolute value of the negative
intercept on the X-axis found by linear regression
analysis of Hyp standards was taken to represent the
Hyp content of the tissue. Quantification of total and
fractionated forms (AS, SS and INS) of collagen was
performed according to the standardized calculation
by Neuman and Logan (1950).
The following formula was used to calculate the
collagen content.
Collagen content = 7.46
¯
[mg of Hydroxyproline per
g freeze-dried sponge weight].
Where, the factor, 7.46 = ratio of the weight of
collagen and weight of hydroxyproline
Note: Collagen content is expressed as mg collagen / g
freeze-dried sponge weight.
1.6 Statistical analyses
All the data in triplicates (n=3) was analyzed using
the student’s
t
-test to quantify macromolecules and
multiple replicates of sponge tissue samples were
used for microscopic observations. Statistical
significance for all the biochemical analyses was
defined as P < 0.05.
2 Results
According to our earlier explorations from the year
2006, there are a total of 40 species were collected in
the Mandapam, Palk Bay and Rameswaram regions of
Gulf of Mannar (GoM) (Table 1). Depending on the
abundance and past pharmacological significance, out
of all the collected marine sponges, four experimental
sponges
Hyattella cribriformis
,
Fasciospongia
cavernosa
,
Callyspongia fibrosa
and
Dysidea fragilis
were compared to bring out their ultrastructural and
biochemical analysis in the current studies.
While collecting, the sponges
Hyattella cribriformis
,
Fasciospongia cavernosa
and
Dysidea fragilis
were
found to be comparatively more fragile and easy to be
ripped off. A possible reason for the fragile nature of
these sponges
could be due to the
lesser
content of
collagen or spongin in the matrix owing to less
amount of strength in the skeletal network,
differentiating them from
Callyspongia fibrosa
, where
the situation is in contrary and they possess greater
amount of collagen matrix.
The morphotypic features
and key characters along with the whole mount
images of these sponges at the time of collection are
presented in the Table 2. The specimen of
Hyattella
cribriformis
is brittle and lightweight, yellowish-white
or pale in color. This sponge is available at around 10
to 15 f from the seashore. Another sponge,
Dysidea
fragilis
,
is available at the depth of 15 to 20 f.
Morphologically, this species appears as a mass of
greenish black to yellow color. Yet another member of
Demospongiae,
but belonging to a different order
family
Callyspongiidae,
Callyspongia fibrosa
,
is
composed of finger shaped or flattened branches,
surface is with strong conules and has prominent
conules at growing tips. The osculae irregularly
distributed, terminal, marginal, rounded or elliptical,
shallow and compound. They appear to be pale yellow
to brown when alive. Dermal skeleton is reticulate,
meshes triangular or subdivided by tertiary fibres,
which are either uni- or multispicular.
2.1 Microscopic Study
Video microscopy
The four sponges showed markedly different skeletal
alignment and distinctive arrangement of bundle of
spicules or oxeae along with the network of spongin,
when observed under video microscope (Figure 1). In
these sponges, the major canals are strengthened with