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International Journal of Marine Science 2013, Vol.3, No.19, 151-157
http://ijms.sophiapublisher.com
155
slightly heating the tip in a flame. Nylon wool fibers
were finely teased using a pair of forceps and the teased
fibers were packed (loosely) into the straw. Adding 5
mL of physiological saline washed the packed nylon
wool column. A small opening was made at the sealed
end of the straw to drain the physiological saline. After
washing with physiological saline, the nylon wool was
then filled with 3 mL of RPMI 1640 medium in a
horizontal position. The nylon wool column was kept
in the incubator (at 37
℃
for 30 minutes) in horizontal
position. This process activates the nylon wool column.
Resuspended lymphocytes were loaded into the
activated nylon wool column. Then the column was
held vertically above an eppendrof tube, now hot saline
(about 60
℃
) was slowly dripped into the column. The
hot saline passing out of the column was collected in
the eppendrof tube, which contain and T lymphocytes.
0.2 mL of the saline containing T lymphocyte (from the
eppendrof tube containing T cell) was taken in a
separate eppendrof tube. To this 0.2 mL of 1% SRBC
was added and then the mixture was centrifuged for 12
minutes at 1600 rpm. After centrifugation the sample
were incubated in an icebox or refrigerator (at 4
℃
) for
5 minutes. After cold incubation, the pellet in the
eppendrof tube was resuspended by gentle flushing
with a Pasteur pipette. Then a drop of it was taken in a
clean dry slide, observed and enumerated T cells under
the microscope (20×/40×) for rosettes. Number of T
cell rosettes formed were observed among hundred
lymphocytes observed was tabulated.
3 Results and Discussion
3.1 Immunity Study in Rats
The effect of the ethyl acetate extracts of
Aurora
globostellata
was tested for their immumunomodulatory
effect in murine models. To test the efficacy of the
extracts of
A. globostellata
, three different does viz;
50 mg/kg, 150mg/kg, 250 mg/kg were used. For
immunological study both cell medicated and
antibody mediated responses were evaluated. To
evaluate the efficacy of the extract to modulate cell
mediated immune (CMI) response, Delayed Type of
Hypersensitivity (DTH) reaction was studied. For
evaluating Antibody mediated immune (AMI) response
Haemoagglutination titer (HA titer) was estimated in
control and sponge extract treated rats.
3.2 Antibody titre
The extracts of
Aurora globostellata
(Ag), (50 mg/kg,
150 mg/kg, and 250 mg/kg) produced a dose related
increase in the primary and secondary antibody
formation with the maximum increase of 120.12±45.6
and 415±202.2 at a dose of 250 mg/kg after 7
th
and
14
th
days respectively. The result indicates that
3-hydroxytetradecanoic acid in the extracts of
Ag
elevated primary and secondary antibody production
significantly (P<0.01). The effect was more in secondary
immune response (Table 1).
3.3 Delayed-type hypersensitivity response (DTH)
The extracts of
Aurora globostellata
(50-250 mg/kg)
increased DTH in Rats (Table 2, Figure 1).
The DTH response, which is a direct correlation of
cell mediated immunity (CMI), was found to be
significantly increased on treatment with sponge
extract. During CMI responses, During CMI responses,
sensitized T-lymphocytes, when challenged by the
antigen, are converted to lymphoblast’s and secrete
lymphokines attracting more scavenger cells to the
site of reaction. The in filtering cells are thus
immobilized to promote defensive (inflammatory)
reaction. Increase in the DTH response indicates the
stimulated effect on lymphocyte and accessory cell
type required for the expression of reaction (Solanki et
al., 2010; Dhasarathan et al., 2010).
Table 1 Effect of the extract of
A. globostellata
on Haemoagglutination (HA titre) assay in Rat challenged with antigen
Animal group
Treatment dose (mg/kg
-1
)
Haemoagglutination (HA titre) log
2
2
7
th
day
14
th
day
Group I
Control
9.60±5.30
24.70±4.80
Group II
Dexamethasone 0.5mg/kg
-1
5.63±0.86
18.00±3.41
Group III
Immun Aid (Pro immune Tablets)
144.00±5.60
256.23±5.10
Group IV
Purified compound T
1
(50mg/kg
-1
)
20.30±4.20
46.15±14.25
Group V
Purified compound T
2
(150mg/kg
-1
)
80.12±18.30
140.10±40.30
Group VI
Purified compound T
3
(250mg/kg
-1
)
120.12±45.60
415.00±0.22
Note: Each data represents the mean ± SE of 6 Wister rats