Contents

Introduction

Objectives

Methodology

Results

Extended proposal for PhD

 Immunomodulator

• A substance of biological or synthetic origin

which can stimulate, suppress or modulate
any of the components of immune system
including both innate and adaptive arms of
immune response. ( Agarwal and Sigh, 1999)
Immunostimulants Immunosuppressant

Immunoadjuvants

Immunomodulators
 Immunomodulatory agents from
natural products

• While several types of synthetic

immunomodulatory agents are available,
undesirable side effects often limit their
use.
• Thus, evaluation of immunomodulatory
activity of natural products provides
better solution for this problem.
 Phyllanthus sp
• Phyllanthus has more than 700 species in at least 11 sub-genera.
(Holm-Nielsen 1979)
• Have long been used in folk medicine to treat treat kidney and
urinary bladder disturbances, intestinal infections, diabetes, and
hepatitis B.(Jayaram and Thyagarajan, 1996)
• P. amarus, P. debilis, P. maderaspatensis, P. virgatus, P. urinaria,
P.niruroides, P. anisolobus, P. orbiculatus, P. emblica, P. oxyphyllus, P.
flexuosus, P. raticulatus,P. fraternes, P. simplex, are among the
species of genus phyllanthus which are commonly used for
medicinal purposes. (Calixto et al., 1998)
 Phytochemical from P.amarus and its
pharmocological properties
• The active phytochemicals, flavonoids, alkaloids, terpenoids,
lignans, polyphenols, tannins, coumarins and saponins, have
been identified. (Ahmad et al., 2003)

• P.amaruspossesses antiviral, antibacterial,

antiplasmodial, anti-inflammatory,
antimalarial, antimicrobial, anticancer,
antidiabetic, hypolipidemic, antioxidant,
hepatoprotective nephroprotective and
diurectic properties(Halliwell et al., 1987)
 Phytochemical from P.urinaria and its
pharmocological properties
• The active phytochemicals, flavonoids, alkaloids, terpenoids,
lignans, polyphenols, coumarins and saponins, have been
identified.(Calixto et al., 1998)

• It has many biological activities, including anti-

hepatitis B virus, anti-Epstein–Barr virus and anti-
retroviral reverse transcriptase, anti-
inflammatory, anti-oxidative, detoxify poison
from body and increase the flow of the urine.
(Chiu et al,1998)
 Objectives
 To isolate and identify the immunomodulators from Phyllanthus amarus and Phyllanthus
urinaria.
 To develop analytical methods for qualitative and quantitative determination of bioactive
compounds in the extracts of Phyllanthus sp.
 To evaluate the mechanism of action of the bioactive isolates of Phyllanthus sp on the
chemotaxis, cell surface glycoprotein expression, phagocytic and respiratory burst activities of
human leukocytes.
 To investigate the mechanistic effects of the bioactive isolates of Phyllanthus species on T-cell
proliferation, nitric oxide production and secretion of pro-inflammatory cytokines in different
lineages of immune cells.
 To develop the qualitative and quantitative chromatographic methods for the preparation of
standardized extracts of Phyllanthus amarus and Tinospora crispa.
 To evaluate the immunomodulatory effects of the standardized extracts of Phyllanthus amarus and
Tinospora crispa on the humoral and cellular components of immune response.
 To investigate the immunomodulatory effects of the standardized extracts of Phyllanthus amarus
and Tinospora crispa on cytokine and lymphocytes levels in innate and adaptive immune response
in vivo.
 Methodology
Content

Extraction and Isolation of P.amarus and P.urinaria

 HPLC standardization and Quantitative analysis

In-vitro Immunomodulatory Assays

• Chemotaxis Assay

•Chemilluminescence Assay

•Phagocytic Assay

•Cd18 surface expression Assay

• Inhibition of T-cell Proliferation Assay

• Cytokine Assay
 Extraction and Isolation of P.amarus
and P.urinaria
Collection of plants
Place of collection : Marang,
Terengganu
Plants were air dried for
The voucher specimens
1 week
(P. amarus UKMB 30078 and P. Extraction of plants (cold maceration)
urinaria UKMB 30077) deposited
at the Herbarium of UKM, Bangi,
Malaysia
MeOH&80% EtOH

Isolation of active
compounds BIOACTIVE EXTRACT Bioassays

Chemilluminescence Assay
Phagotest assay T-cell proliferation assay

Cytokine assay
Chemotaxis assay
CD 18 surface
glycoprotein expression
assay
 Extraction and Isolation of Phyllanthin and
Hypophyllanthin (marker compounds).

The plant materials were allowed to dry under shade.

500 g of dried material of each plant sample were

ground and macerated in methanol Phyllanthin Hypophyllanthin

Extracts of P.amarus (55.2g, 11.04%w/w) and P.urinaria

(52.7g,10.54%w/w) were obtained Structural determination by using NMR
spectroscopy

10g of extract of P.amarus were subjected to VLC

( Silica type-H),
Gradient elution with : Phyllanthin ( 228.5mg)
Hex: ChCl3 (10:0-9:9v/v) Hypophyllanthin ( 321.2mg)
CHCl : MeOH (10:0-0:10) Purity determined by using HPLC

Repeated chromatography (silica gel 40×63µm, 3×60cm)

Eluted with gradient system Crystals were purified
N-Hexane : EtAC (10:0-1:9,v/v) with hexane
HPLC analysis of extracts of Phyllanthus amarus and Phyllanthus
urinaria
Instrument method
Sample preparation
Instrument : Waters HPLC
Column: Reverse Phase, C-18 column (250mm × 4.6mm
i.d., 5 𝜇m, Xbridge, Waters, Ireland)
Extracts/Standards dissolved
Method 1
in HPLC-grade methanol at
Flow rate : 0.4 mL /min
concentration Mobile phase :
20 mg/ml (extracts) Solvent A : 0.1% Trifluoroacetic acid in mili-Q water
1000µg/ml- 32µg/ml Solvent B : Acetonitrile
(standards) Isocratic 5% B for 5 minutes, then increasing to 95% B over 20
minutes. Hold at 95% for minutes
Detection wavelength : 205nm,229 nm, 254nm

Method 2
Flow rate : 0.6ml/min
Mobile phase
Solvent A : 0.1% Trifluoroacetic acid in mili-Q water
Solvent B : Acetonitrile
Procedure :5-70% A ( 0 to 15min), 70 to 95% A ( 15 to 30min) and
pump A had been hold at 95% for twenty minutes. Whereas
gradient elution was performed with 5% B which was then
increased to 95% over 20 min and had been hold at 95%for 15min
for analysis of extract
Detection :wavelength : 270nm
In-Vitro Immunomodulatory
Bioassays
 Viability assay
Cytotoxicity of P.amarus and P.urinaria extracts and compounds were
measured by using MTT- reduction method for MNCs, while for PMNs,
Cell viability was determined by the standard trypan blue exclusion method.
For MTT assay,100µl sample were filled into a 96 well round bottom
microplate containing 100µl cells suspension in RPMI 1640 10% FCS.
The plates were incubated for 24 hours at 37ºC and 5% CO2. 20µl of MTT
reagent (1mg/ml) was added into each well and incubated for 4 hours at
37ºC and 5% CO2. The supernatant removed and the formazan produced by
cells in each well was dissolved in DMSO and the optical densities (OD)
were measured by ELISA readers at wavelength 570nm .

CHEMILUMINESCENCE ASSAY
25μL PMNs or
MNCs
25μL of sample / aspirin
Incubated for 30 minutes at 37°C
25μL Luminol / + 25μL serum
opsonized zymosan / PMA were added to
each well and final volume adjusted
to 200μl by HBSS ++.
Results were monitored
as chemiluminescence
RLU by luminometer
Chemotaxis assay
25µl fMLP +
25µl samples
were added to
lower chamber
25 µl
PMN/MNCs
cells were added
to upper chamber
Incubated for 1 h at
37°C in CO2 incubator
Membrane
filter was fixed
Membrane filter was
removed and stained with
hematoxylin.
Migration of cell observed
and measured using
microscope
CD18 surface expression Assay
Phagocytosis Assay

100µl of blood
100µl of peripheral blood +
aliquot into
1µl of LPS (0.25µg/ml) + 20µl
falcon tube
of sample / aspirin and
without sample / aspirin for
negative control
Sample added
into falcon tube

Incubated for 90 minutes at Finally 500µl of PBS

added and measured Incubated at 37C
37°C in CO2 incubator water bath for 10
by using flow
cytometer minutes
Tubes were incubated in ice box
to stop reaction
5µl of E.coli was
Supernatant was added
10µl CD18-FITC / 10µl removed and
Immunoglobulin G -FITC washed with 3ml of
PBS × 2 100 µl of icecold
QUENCHING SOLUTION will
Incubated in ice for 60 be added
minutes
Centrifuged at
250g for 5 Cells were washed and lysing
minutes at 4°C buffer was added
FACS lysing solution was added and
incubated at room temperature for 20 Measured
minutes using flow
DNA solution was added cytometry
 T-cell proliferation assay

Sample or compound
50µl of 5% RPMI medium was
was added into the well 50µl of PBMC cells were
added into each well of 96 well
except the control well added into each well.
round bottom plate
Except the blank

5µl of [H]3 Thymidine was added

Incubated for 72 hours in CO2 50µl 5% complete medium
and incubated in CO2 incubater at
incubater at 37 C of RPMI to top it up to
37C for 18 hours
0.2ml

Reading was taken from

scintillation counter .

CYTOKINE ASSAY
LPS + plant sample
will be added to the
cell fraction in 96
wells microtiter
plates.
Covered with adhesive strip and
incubated at 37, 5% CO2 for
several hours depending on the
level of cytokines
The assessment of cytokines levels in
supernatants of human blood cell cultures will
be accomplished using the appropriate ELISA-
kit

The cytokine –concentration in the sample

will be determined from appropriate
calibration curves.
Results
Standardization and Quantification analysis of Lignans found in 80% Ethanol
extracts of Phyllanthus amarus and Phyllanthus urinaria

Method 1
1
2

Figure 1: Representative HPLC chromatograms of (a) P.amarus , (b) P. urinaria for identification
and quantification of Phyllanthin(1) at RT 27.251minutes, Hypophyllanthin (2) at RT 28.079
minutes.
 Standardization and Quantification analysis of Phenolic compounds
found in 80% Ethanol extracts of Phyllanthus amarus and Phyllanthus
urinaria

Method 2
AU

a
AU

MINUTES

Figure 2: Representative HPLC chromatograms of (a) P.amarus , (b) P. urinaria for identification
and quantification of gallic acid (1) at RT 8.172 minutes, geraniin (2) at RT 23.694minutes,
corilagin (3) at RT 26.436 minutes, ellagic acid (4) at RT 33.529minutes.
 HPLC Quantification analysis data
Table 1 : Amount of major compounds found in P.amarus and P.urinaria (µg/ml)
obtained from HPLC quantification analysis.

Ellagic
Species Phyllanthin Hypophyllanthin Gallic acid acid Corilagin Geraniin

Phyllanthus amarus 103.50 44.00 163.30 601.29 313.41 170.49

Phyllanthus urinaria 16.80 9.50 103.48 231.34 159.37 173.17

 In-vitro
immunomodulatory assays
 Inhibition of PMN& MNCs migration by sample
chemotaxis of PMNs
Inhibition (%) of
of MNCs
of chemotaxis
Inhibition (%)

Sample

Figure 3: Percentage of inhibition of Phyllanthus sp. and their major compounds on PMN and MNC
chemotaxis. Data are mean ± SEM (𝑛 =3). Significance of differences with respective control: *𝑃 <
0.05.
Chemilluminescence assay
from PMNs
Inhibition (%) of ROS generation
from MNCs
Inhibition (%) of ROS generation

Sample

Figure 4: Percentage of inhibition of ROS inhibitory activity of Phyllanthus sp. and their
major compounds on PMA stimulated PMNs (a) and MNCs (b) assayed by luminol
amplified chemiluminescence. Data are mean ± SEM (𝑛 = 3). Significance of
differences with respective control:*𝑃 < 0.05.
from PMNs
Inhibition (%) of ROS generation
from MNCs
Inhibition (%) of ROS generation

Figure 5: Percentage of ROS inhibitory activity of Phyllanthus sp. and their major
compounds on Zymosan stimulated PMNs (a) and MNCs (b) assayed by luminol
amplified chemiluminescence. Data are mean ± SEM (𝑛 = 3). Significance of
differences with respective control:*𝑃 < 0.05.
 TABLE 3: IC50 values (𝜇g/mL) of ROS inhibitory and chemotaxis activities of Phyllanthus sp.
and their major compounds on human blood cells (Mean± SEM, 𝑛 = 3). IC50 values in 𝜇M are
in parentheses.

IC50 Values (µg/mL)

Chemotaxis Chemiluminescence
fMLP Zymosan PMA
Sample PMNs MNCs Whole Blood PMNs MNCs Whole Blood PMNs MNCs
PA (Mal) 1.22 ± 0.10 1.44 ± 0.80 0.95 ± 0.24 0.58 ± 1.80 0.10 ± 0.17 1.24 ± 0.98 0.42 ± 0.75 0.56 ± 0.30
PU (Mal) 1.46 ± 0.10 2.84 ± 0.30 1.52 ± 0.53 1.00 ± 1.21 1.23 ± 0.88 1.98 ± 1.09 0.45 ± 0.23 0.26 ± 0.12

GA 1.40 ± 0.02 2.00 ± 0.03 1.70 ± 0.03 0.43 ± 1.02 1.29 ± 0.87 0.87 ± 0.12 0.48 ± 0.01 1.28 ± 0.01
(7.66±0.15µM) (11.15±0.23µM) (9.99±0.12µM) (2.53±0.10µM) (7.58±0.50µM) (5.11±0.15µM) (2.82±0.15µM) (7.52±0.12µM)
EA 1.33 ± 0.07 1.81 ± 0.12 1.36 ± 0.08 0.56 ± 0.02 0.45 ± 0.09 0.83 ± 0.03 0.31 ± 0.07 0.75 ± 0.12
(4.40±0.12µM) (5.96±0.12µM) (4.50±0.16µM) (1.85±0.35µM) (1.49±0.07µM) (2.78±0.07µM) (1.02±0.06µM) (2.4 ±0.06µM)
Ger 1.04 ± 0.12 1.61 ± 0.14 0.34 ± 0.30 0.18 ± 0.07 0.37 ± 0.41 0.19 ± 0.12 0.12 ± 0.04 0.36 ± 0.61
(1.09±0.23µM) (1.69±0.05µM) (0.36±0.12µM) (0.18±0.02µM) (0.38±0.07µM) (0.21±0.01µM) (0.13±0.01µM) (0.38±0.12µM)
Cor 1.49 ± 0.024 1.99 ± 0.09 0.37 ± 0.37 0.36 ± 0.07 0.44 ± 0.08 0.22 ± 0.88 0.15 ± 0.04 0.44 ± 0.03
(2.34±0.07µM) (3.13±0.44µM) (0.58±0.03µM) (0.56±0.023µM) (0.69±0.12µM) (0.35±0.085µM) (0.24±0.02µM) (0.69±0.05µM)
Ibuprofen 1.40 ± 0.10 1.98 ± 0.05
(6.60±0.10 µM) (9.57±0.23µM)

Aspirin 2.16 ± 0.80 0.85 ± 0.20 0.43 ± 0.07 0.17 ± 0.01 0.14 ± 0.09 0.58 ± 0.28

(11.88±4.14µM) (4.68±2.71µM) (2.40±0.16µM) (0.94±0.05µM) (0.77±0.16µM) (3.22±0.38µM)

Phyll 3.20 ± 0.15 8.51 ± 0.52 3.70 ± 2.80 3.2 ± 2.81 2.38 ± 1.44 1.40 ± 1.72 0.08±0.66 1.54±0.87
(7.60 ±0.10µM) (20.2±0.40µM) (8.70 ± 0.60µM) (8.10 ± 3.40µM) (5.68±0.31µM) (3.34±0.05µM) (0.20±0.01µM) (3.68±0.43µM)
Hypophyll 4.40 ± 0.2 9.60 ± 0.26 4.00 ± 1.40 5.00 ± 2.32 1.41 ± 0.06 1.87 ± 0.01 0.95 ± 0.11 1.73 ± 0.31
(11.5 ± 4.70
(10.3 ±0.40 µM) (22.3±0.53µM) (9.20 ± 3.30µM) (3.27±0.15µM) (4.34±0.02µM) (2.20±0.31µM) (4.02±0.71µM)
µM)
 TABLE 4 : Percentage of phagocytic activity (%) of neutrophils and monocytes at
various concentrations of Phyllanthus extracts and their major compounds. (Mean ±
SEM, 𝑛 = 3).

Sample Inhibition (%)

Concentration (µg/ml)
PMNs MNC PMN MNC PMN MNC PMN MNC
100µg/ml 50µg/ml 62.5µg/ml 31.25µg/ml
PA (Mal) 74.20 ±1.17 56.68 ± 0.82 79.40 ± 1.23 60.70 ± 0.15
PU (Mal) 84.70 ± 0.98 60.80 ± 0.11 78.53 ± 2.07 66.08 ± 1.66
GA 95.90 ± 1.52 77.80 ± 2.18 80.60 ± 1.97 61.80 ± 1.84
EA 98.18 ± 1.19 75.50 ± 1.97 85.20 ± 2.13 63.10 ± 2.31
Ger 97.10 ± 3.05 80.28 ± 2.06 86.80 ± 1.06 64.90 ± 0.91
Cor 99.30 ± 2.82 82.70 ± 1.51 85.50 ± 0.32 63.30 ± 1.04
Phyll 61.72 ± 1.39 48.59 ± 0.84 72.81 ± 2.03 53.86 ± 1.35
Hypophyll 70.25 ± 0.53 54.77 ± 0.63 22.33 ± 0.29 59.70 ± 1.07
Positive control 87.80 ± 1.25 65.20 ± 0.36
 TABLE 5 : Percentage of CD18 expression (%) on neutrophils, monocytes and
lymphocytes at various concentrations of Phyllanthus extracts and their major compounds.
(Mean ± SEM, 𝑛 = 3).

Sample Inhibition (%)

Concentration (µg/ml)
PMNs Lymphocytes MNCs PMNs Lymphocytes MNCs
100µg/ml 50µg/ml
PA (mal) 93.61 ± 1.70 84.84 ± 0.12 90.99 ± 2.13
PU (mal) 89.90 ± 1.08 72.33 ± 2.35 86.20 ± 1.54
GA 90.77 ± 1.80 76.84 ± 2.20 90.70 ± 1.20
EA 93.13 ± 2.31 78.64 ± 2.05 91.59 ± 0.57
Ger 96.97 ± 1.71 81.60 ± 1.80 84.49 ± 1.71
Cor 98.80 ± 0.24 85.80 ± 0.61 88.76 ± 1.10
Phyll 67.17 ± 1.34 50.80 ± 0.12 61.56 ± 0.98
Hypophyll 74.70 ± 0.09 58.90 ± 0.97 74.42 ± 0.77
Positive control 86.77 ± 0.54 75.84 ± 0.76 84.79 ± 0.55
 IC50 Values (µg/mL)
Inhibition of Nitric oxide Inhibition of T-cell proliferation Inhibition of cytokine release
IL-1B TNF-alpha
Sample
PA (Mal) 3.69±0.45 2.57 ±1.36 6.62±0.23 4.918±0.71

PU (Mal) 5.58±0.87 9.31 ±1.27 3.59±0.57 9.484±1.44

GA 4.12±0.32 0.96.32±0.06 8.76±0.69 17.72±0.75

(24.17±1.37µM) (5.65±0.34µM) (5.14±1.87µM) (104.16±1.35µM)
EA 5.548±0.22 13.23±1.24 16.20±0.61 20.44±1.66
(18.36±1.09µM) (43.77±1.54µM) (53.61±1.78µM) (67.64±2.03µM)
Ger 4.25±0.47 12.31±1.68 19.82±2.56 7.398±1.31
(7.81±0.31µM) (22.6±1.32µM) (36.4±5.57µM) (13.59±2.56µM)
Cor 5.09±1.75 11.08±1.17 27.40±1.17 13.92±0.97
(7.98±2.05µM) (17.41±2.12µM) (43.05±2.08µM) (21.87±1.89µM)
Phyll 0.76±0.62 14.59±0.06 2.10±0.98 17.779±0.75
(1.82±1.13µM) (34.86±0.11µM) (5.02±1.36µM) (40.58±1.44µM)
Hypophyll 0.93±3.45 19.09±0.83 8.38±0.14 12.11±0.77
(2.18±5.25µM) (44.37±2.09µM) (19.48±0.88µM) (29.44±1.39µM)
Prednisolone 0..0411±0.99 0.26±0.12 0.77±1.98
(0.09±1.28µM) (0.67±1.27µM) (1.96±2.11µM)

Dexamethasone 0.012±1.17
(0.02±2.46µM)

IC50 values (𝜇g/mL) of NO inhibitory, inhibition of T-cell proliferation and inhibition of

proinflammatory cytokinesiactivities of Phyllanthus sp. and their major compounds on
phagocytes (Mean± SEM, 𝑛 = 3). IC50 values in 𝜇M are in parentheses
In vivo immunomodulatory effect of
80% ethanol extract of Phyllanthus
amarus in Wistar-kyoto (WKY) rats
Evaluation of Innate Immune Response

 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
 Evaluation of Cellular Immune Response

CD 11b/ CD18 surface antigen expression assay.

Leukocyte migration assay- isolated PMNs.

Phagocytosis Assay- Isolated PMNs .

T and B cell proliferation

Measurements of B and T-lymphocyte sub populations

Macrophage activity on phagocytosis of E.coli

Nitric oxide production from peritoneal macrophage

 Wistar Kyoto rats (Total 108)

Allow at least 1 week to adapt to laboratory

environment

Rats will divided in groups with 6 in each groups

-Ve Control +Ve Control

normal saline Test Group
Cyclophosphamide

100 mg/kg extract 200 mg/kg extract 400mg/kg extract

 Immunization schedule
 Receive the extract doses of 100,
200 and 400 mg/kg P.O.

 Rats challenged on day 14th day.

 15th day blood collected by retro-

orbital plexus.

 21st day rats euthanized and

collected organs.

Immunization : 20% of SRBC ( 5 × 109 SRBC/rats)

 Doses of extracts and standards
 Doses of extracts and standards are selected based on
literature review

 Doses of Phyllanthus amarus/ Tinospora crispa : 100, 200

and 400mg/kg p.o respectively. (Oyewo, Bukoye E, Akanji,
Musbau A et al.,2014)

 Dose of Levamisole : Levamisole 10 mg/kg was used as the

reference immunostimulant . (George A, Koffuor, Patrick A
et al., 2011)

 Cyclophosphamide : 15mg/kg of body weight of rats ( 14

days) oral (Hou, Yang et al.,2007)
 Measurement of neutrophil transmigration

Quantitatively accessed by using 24-well Cell Migration Assay kits

1.5 × 106 cells in 300 μL serum-free media were added to the upper chamber
in 24-well tissue culture plate

500 μL RPMI media containing 10% FBS as a chemoattractant was added to

the lower chambers of the 24-well plate.

Incubated for 2.5 h at 37 ◦C and 10% CO2.

cThe number of cells migrating to lower chambers was determined by

fluorescence
 B/ T Lymphocyte proliferation assay

Spleen was excised and minced to collect splenocytes

Cells were cultured (100 ul, 4 x 105 cells per well) in triplicate with an equal
volume of mitogen ( PHA (20µg/ml), LPS ( 10µg/ml) preparation

Incubated at 37°C in a humidified atmosphere with 5% CO2 for 48 h.

[methyl-3H]thymidine (0.5 ,uCi/well)

Uptake of [3H]thymidine evaluated with a liquid scintillation counter

 Flow cytometry of lymphocyte sub-
population
Splenocytes incubated with FITC labeled anti-rat CD3, CD4 and
PE conjugated anti rats CD8 antibodies.

After incubation, cell will be washed and fixed in 0.5%

paraformaldehyde

Analyzed by flowcytometer

• Cytotoxic T-cells (CTLs), which have CD8 present on their surface (CD8+ T-cells)

• Helper T-cells, which have CD4 present on their surface (CD4+ T-cells)
Measurement of Mac-1 upregulation by flow cytometry

neutrophils wil be stimulated with fMLP (1 mM) for 15 min

Cells pelleted and resuspended in 1 ml ice-cold PBS containing 10% heat-

inactivated foetal bovine serum (FBS) and10 mM sodium azide.

Incubated for 60 min with fluorescein isothiocyanate (FITC)-conjugated anti-

Mac-1 antibody (mouse anti-rats) CD11b or CD18, class IgG1

Washed with PBS containing 5% FBS, stained cells were resuspended in flow
cytometer sheath fluid containing 1% of paraformaldehyde.

Mac-1 expression measured using flow cytometry

Shen et al., 1999
 Measurement of Th1/Th2 cytokines

 Sepration of serum from blood

 Th1 cytokines
 TNF-α, IL-2 and IFN-γ

 Th2 cytokines
 IL-4

Quatitaively determined by 5 plexmagnetic beads based

ProcartaPlex rat immunoassay kit (eBioscience).
 Toxicity Evaluation of P.amarus extract

Parameters Doses of extracts (mg/Kg)

Control Vehicle 100mg/Kg 200mg/Kg 400mg/Kg

Initial body weight (g) 309 ± 2.13 315 ± 1.13 309 ± 2.15 311 ± 0.88 312 ± 1.87

Final body weight (g) 324 ± 1.54 335 ± 0.89 328 ± 1.97 332 ± 2.76 338 ±1.88

Spleen (g) 0.61 ± 3.87 0.65 ± 1.14 0.74 ± 1.82 0.68 ± 2.11 0.72 ± 1.92

spleen /body weight 4.46 ± 1.06 3.25 ± 0.98 3.89 ± 0.57 3.23 ± 0.21 2.76± 0.42
 PMN Migration Assay
250
y = 0.0009x + 18.551

200
Relative fluorescence unit

150

100

50

0
0 50000 100000 150000 200000

No of cells
 Lymphocyte proliferation

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
Th1/Th2 cytokine release assay
Evaluation of Adaptive Immune Response

 Estimation of serum level IgG and IgM

using ELISA kits
 Serum level of ceruloplasmin and
Lysozyme
 MPO activity of liver tissue homogenate
 Macrophage activity on phagocytosis of
E.coli
 Nitric oxide production from peritoneal
macrophage
 Balb C mice (male)

Allow at least 1 week to adapt to laboratory

environment

Animals divided in groups with 6 in each groups

-Ve Control +Ve Control

normal saline Test Group Levamisol
Cyclophosphamide

50 mg/kg extract 100 mg/kg extract 200mg/kg extract

 Immunization schedule

Mice were immunized on day 0

of treatment.

Immunized group were

challenged on day 7th

14th day mice euthanized and

collected organs.

Immunization : 20% of SRBC ( 5 × 109 SRBC/rats)

Analysis of IgG and IgM antibody Lysozyme and
Ceruloplasmin

Blood collected from animals on 7th and 14th days after challenging to
obtain serum

The levels of IgG and IgM antibody Lysozyme and Ceruloplasmin

determined by ELISA

The assay performed using protocol given by manufacturers

 Phagocytosis of macrophages

20µl of chilled E.coli added into 5ml tubes containing 100µl of

macrophage.

The tubes were shaken at low speed for 3 s and experimental samples
kept at 37C, and control sample at 0C

Quenching solution added into sample tubes washes and centrifuged

200µl of DNA solution was added. The test determines phagocytizing

macrophages
Nitric oxide production from peritoneal macrophages

SRBC immunized mice administered with extract or standard control for 14 days

Sacrificed mice injected with 10ml ice cold RPMI medium (i.p). Macrophages
were collected peritoneally after lavaging mice

The tubes centrifuged at 1800rpm for 10 min at 4C. The supernatant was thrown
and pellet dissolved in RBC lysis buffer. Tubes were incubated in ice for 5 min.

The tubes centrifuged at 1800rpm for 10 min at 4C. The supernatant was thrown
and pellet dissolved in 1ml of RPMI medium.

Cell viability determined using tryphan blue. The cell were cultured in U bottomed
96 well plate. The plate was incubated in CO2 chamber for 24 hours.
 Serum Lysozyme Levels

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on serum
level lysozyme release in splenocytes of experimental rats.
Notes: Results are represented as mean ± SEM, with n=6 in each group.
**P<0.01; *The statistical tests employed were ANOVA, followed by post-
Dunnett’s test.
 Serum Ceruloplasmin Levels

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on serum level
ceruloplasmin release in splenocytes of experimental rats.
Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01; *The
statistical tests employed were ANOVA, followed by post-Dunnett’s test.
 Serum IgG Levels

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on serum level IgG
release in splenocytes of experimental rats.
Notes: Results are represented as mean ± SEM, with n=6 in each group. *P<0.05; The
statistical tests employed were ANOVA, followed by post-Dunnett’s test.
 Serum IgM Levels

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on serum level IgM
release in splenocytes of experimental rats.
Notes: Results are represented as mean ± SEM, with n=6 in each group. *P<0.05; The
statistical tests employed were ANOVA, followed by post-Dunnett’s test.
 Phagocytosis of E.Coli by Macrophages

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on phagocytosis of
E.coli by macrophages
Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01; *The
statistical tests employed were ANOVA, followed by post-Dunnett’s test.
Flowcytometric Evaluation
Nitric oxide production from peritoneal macrophage

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on nitric oxide
release by macrophages
Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01;
*The statistical tests employed were ANOVA, followed by post-Dunnett’s test.
 MPO activity of Liver tissue Homogenate

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on MPO release.
Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01;
*The statistical tests employed were ANOVA, followed by post-Dunnett’s test.
 Delayed Type hypersensitivity

Effect of Phyllanthus amarus extract on DTH assay

Groups

Paw thickness (mm)

Before challenge After challenge

Sensitized
Control 0.276±0.21 0.284± 0.55

Phyll-20mg/Kg 0.180±0.11 0.287± 0.48

Phyll-40mg/Kg 0.194±1.03 0.287± 0.29

Phyll-100mg/Kg 0.208±0.98 0.280± 0.99

Cylophosphamide 0.187±0.77 0.431±1.01

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 With immunization

Negative Test group Positive control

control group (ii) will Negative Test group Positive control
receive group (2ii) will
group(i) will Group (iii) control
cyclophasphamide receive
(receive group(2i) will Group (2iii) cyclophasphamide
saline water (receive
solution) saline water
solution)
group (iiia), (iiib) and( iiic)

( receive phyllanthin only) 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.

Relative organ weight

N Body weight of mice (g) (mg/g)
Initial 7 14 Spleen Liver
Negative control 22±0.98 24 24 4.833 46.29
20mg/Kg 24±1.01 24 23 5.21 59.01
40mg/Kg 21±0.56 23 23 4.96 48.48
100mg/Kg 21 22 22 6.636 50.01
Cyclophosphamide-
200mg/Kg 23 19 20 4.03 45.13
Effect of phyllanthin on PMNs isolated from treated Balb/C
mice

Cyclophosphamide-100mg/kg Phyllanthin 10mg/kg

count

E.Coli FITC-A
control Phyllanthin 100mg/kg
 Effect of phyllanthin on PMNs isolated from treated Balb/C
mice

Evaluation of the effect of phyllanthin(20, 40, 100 mg/kg) on Ecoli engulfment by

macrophages .
Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01;
*P<0.05. The statistical tests employed were ANOVA, followed by post-Dunnett’s test.
Effect of phyllanthin on PMNs isolated from treated Balb/C
mice

control Phyllanthin-20mg/Kg Phyllanthin-100mg/Kg

cyclophosphamide Phyllanthin-40mg/Kg
The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
Effect of phyllanthin on CD18/CD11b expression on PMNs

Negative Phyll 20 mg/Kg Phyll 100mg/Kg

Cyclophosphamide Phyll 40mg/Kg

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
Effect of phyllanthin on CD4/CD8 expression on splenocytes

Control Phyll-20mg/Kg

Cyclophosphamide Phyll-40mg/Kg Phyll-100mg/Kg

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
 Effect of phyllanthin on lysozyme/
ceruloplasmin

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
 Effect of phyllanthin on nitric oxide production

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
 Effect of phyllanthin on serum level igG and igM

The values are expressed as mean±SEM with n=6, **P<0.01, *P<0.05, followed by
post-dunnett’s test
 Delayed type hypersensitivity

Effect of Phyllanthus amarus extract on DTH assay

Groups
Paw thickness (mm)

Before challenge After challenge

Sensitized
Control 0.292±0.213 0.311± 0.21

Phyll-20mg/Kg 0.217±0.112 0.412± 0.32

Phyll-40mg/Kg 0.264±1.23 0.489± 0.48

Phyll-100mg/Kg 0.308±0.98 0.515± 0.78

Cylophosphamide 0.276±0.77 0.632±0.98

Thank you