EVALUATION OF IMMUNOMODULATORY
POTENTIAL OF PLANT EXTRACT(S) IN EXPERIMENTAL ANIMALS
1.0 INTRODUCTION:
Immunomodulation is a process, which alters the
immune system of an organism by interfering with its functions. This
interference results in either immunostimulation or immunosupression. An
immunomodulator is substance that helps to regulate the immune system. This
‘regulation’ is a normalization process, so that an immunomodulator helps to
optimize immune response. Immunomodulators are becoming very popular in the
worldwide natural health were as these do not tend to boost immunity, but to
normalize it. Immune regulation is a complex balance between regulatory and
effector cells and any imbalance in immunological mechanism can lead to
pathogenesis.
2.0 TEST SYSTEM DETAILS:
Species
: Mus musculus (Mouse)
Strain : Swiss albino
Age : 6-8 weeks
Body
Wight : 20-25 g
Sex : Male or Female
No. of
animals : 8+8 /Group
3.0 ALLOCATION OF GROUPS:
|
Groups |
Treatment |
Dose; ROA |
No. of Animals# |
|
G1 |
Normal
Control |
Normal
saline or 0.25% Na-CMC |
8+8 |
|
G2 |
Reference
Drug- Levamisole |
2.5
mpk; p.o. |
8+8 |
|
G3 |
Plant
Extract-1 |
X
mpk; p.o. |
8+8 |
|
G4 |
Plant
Extract-2 |
XX
mpk; p.o. |
8+8 |
|
G5 |
Plant
Extract-3 |
XXX
mpk; p.o. |
8 |
*The dose and ROA (Routes of administration) will be decided
based on the type of reference drug
# One set of animal will be utilized for Delayed Type
Hypersensitivity Reaction assay and another set will be used for the humoral
antibody response assay
4.0 METHODOLOGY:
·
The study protocol (Form B)
shall be approved from the IAEC before commencing the experiment.
·
Animals shall be procured from
the CPCSEA authorized vendor.
·
Animals shall be quarantined
for 1 week as per the in house SOP.
·
Healthy animals will be
selected, randomized based on body weight and divided into five different
groups consisting of 8+8 animals each.
·
Treatment will be given as
follows till day 7 for DTH model and day 10 for Humoral Antibody Response
model.
o Group
G1 animal will be treated as normal control and treated with normal saline or
Na- CMC.
o Animals
of group G2 will be treated orally with Levamisole at the dose of 2.5mpk.
o Group
G3, G4 and G5 animals will be treated with plant extract(s) at different dose
levels.
·
Antigen
preparation
o Fresh
blood will be collected from healthy sheep and mixed with sterile Alsever’s
solution (1:1).
o The
blood will be then centrifuged at 1600×g for 5 min to enable red blood
cells to settle at the bottom of the test tube.
o The
supernatant will be discarded, leaving sheep red blood cells (SRBC) pellets
that will be washed three times with pyrogen-free phosphate buffered saline (pH
7.2).
o They
will be then kept under refrigeration for use in the immunization and challenge
study.
·
Delayed
Type Hypersensitivity Reaction (Cellular Immunity)
o Mice
will be sensitized with 0.1mL of SRBCs suspension containing 1X108
cells, intraperitoneally on day 0.
o The
animals will be challenged seven days later by injecting the same amount of
SRBC into the left hind footpad.
o The
thickness of the left hind footpad was measured with a digital vernier caliper
at 4 h and 24 h after the challenge.
o The
change in paw size is calculated by the formula: Paw size after challenge- Paw
size before challenge
·
Humoral
Antibody Response
o The
mice will be immunized by injecting 0.5 ml of 1X108 cells SRBCs
intraperitoneally (i.p.) on the day of the immunization.
o Blood
samples will be collected by retro-orbital puncture on the 10th day
after the immunization.
o Antibody
levels will be determined by the hemagglutination technique.
o The
antibody titer will be determined by a two-fold serial dilution of one volume
(100 µl) of serum and one volume (100 µl) of 0.1% bovine serum albumin (BSA) in
saline.
o One
volume (100 µl) of 0.1 % SRBCs in BSA in saline will be added and the tubes
will be mixed thoroughly.
o They
will be allowed to settle at room temperature for about 60-90 min until the
control tube showed a negative pattern (a small button formation).
o The
value of the highest serum dilution showing visible hemagglutination will be
taken as the antibody titer.
5.0 END POINT PARAMETER(S):
·
Clinical signs
·
Body weight
·
DTH Response
·
Humoral antibody response
(Antibody titer)
6.0 REFERENCE(S):
6.1 Surendra
S Agrawal, Soirabh C Khadase, Gokul S Talele. Studies on immunomodularoy
activity of Capparis zeylanica leaf extracts. International J Pharma Sci. and
Nanotechnology. 2010; 3(1); 887-892.
6.2 Prashant
Singh Chauhan, Kuldeep Kumar Gupta, Sarang Bani. The immunosuppressive effects
of Agyrolobium roseum and pinitol in experimental animals. International
Immunopharmacology 11 (2011) 286–291.
6.3 SY
Gabhe, PA Tatke, TA Khan. Evaluation of the immunomodulatory activity of the
methanol extract of extract of extract of Ficus benghalensis Ficus benghalensis
Ficus benghalensis roots in rats. Indian J Pharmacol; August 2006; Vol 38;
Issue 4; 271-275.
END OF DOCUMENT
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