The Immunosuppressive Effects of
of Phyllanthin in Balb/c mice
Balb/C mice
Allow at least 1 week to adapt to laboratory
environment
10 groups of rats will be formed with 6 in each groups
Without immunization
Negative
control
group(i) will
(receive
saline water
solution)
Test group
Group (iii)
group (iiia), (iiib) and( iiic)
( receive phyllanthin only)
With immunization
Positive control
group (ii) will
receive
cyclophasphamide
Negative
control
group(2i) will
(receive
saline water
solution)
Test group
Group (2iii)
Positive control
group (2ii) will
receive
cyclophasphamide
group (2iiia), (2iiib) and(2 iiic)
( receive phyllanthin only)
Immunization schedule
Animal groups
Immune response study
- Receive the extracts dose 20, 40 and 80 mg/kg i.p.
- Rats will be challenged on day 14th day.
-15th day blood collected for assessment of immune response.
-21st day rats will be humanized blood collected for humoral immunity
assessment organs will be removed for toxicology studies.
Immunization : 20% of SRBC ( 5 × 109 SRBC/rats)
Assessment of immune functions
• Measurement of IgG and IgM antibodies-ELISA- serum
sample
• Phagocytosis Assay- Isolated PMNs
• Th1/Th2 cytokine releasing Assay – serum sample
• Leukocyte migration assay- isolated PMNs
• CD 11b/18 surface antigen expression assay
• T-cell proliferation Assay using spleen cell suspension
• Measurements of B and T-lymphocyte sub populations
• Myeloperoxidase Assay
Assessment of immune functions
• Delayed Type Hypersensitivity
• Nitric oxide production from macrophages
• Ceruloplasmin and lysozyme analysis
Toxicology Effect of Phyllanthin on body weight and relative
organ weight of Balb/C mice.
N
Negative control
20mg/Kg
40mg/Kg
100mg/Kg
Cyclophosphamide200mg/Kg
Initial
22±0.98
24±1.01
21±0.56
21±0.77
23±1.21
Body weight of mice (g)
7 days
14 days
24±1.04
24±0.32
24±0.82
23±0.88
23±0.97
23±0.67
22±1.11
22±0.55
19±1.09
20±1.32
Relative organ weight
(mg/g)
Spleen
Liver
4.833±1.42 46.29±0.58
5.21±0.89 59.01±0.91
4.96±0.71 48.48±1.21
6.636±0.77 50.01±1.56
4.03±0.66
45.13±1.07
Effect of phyllanthin on macrophages isolated from treated
Balb/C mice
control
cyclophosphamide
Phyllanthin-20mg/Kg
Phyllanthin-40mg/Kg
Phyllanthin-100mg/Kg
Effect of phyllanthin on CD18/CD11b expression on PMNs
Negative
Cyclophosphamide
Phyll 20 mg/Kg
Phyll 40mg/Kg
Phyll 100mg/Kg
Effect of phyllanthin on CD4/CD8 expression on splenocytes
Control
Cyclophosphamide
Phyll-20mg/Kg
Phyll-40mg/Kg
Phyll-100mg/Kg
Effect of phyllanthin on lysozyme/
ceruloplasmin
Effect of phyllanthin on nitric oxide production
Effect of phyllanthin on serum level igG and
igM
Delayed type hypersensitivity