B. Tech Biotechnology Dissertation - SRM University - May 2007

Vivek Krishnakumar (10903253)
School of Biotechnology
SRM University
Cell Biology Lab,
Pharmacology Division,
Indian Institute of Integrative Medicine (CSIR)
Jammu Tawi
15/05/2007
Immunomodulatory Activity of
Silymarin and its Effect on Stress
Advisors
Dr. Sarang Bani (Scientist, IIIM, Jammu Tawi)
Dr. R. S. David Paul Raj (Asst. Professor, SRM University)

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15/05/2007
OVERVIEW
1. Introduction
2. Motivation
3. Objectives of work
4. Immunomodulatory study
a. Protocol
b. Results
5. Stress related study
a. Protocol
b. Results
6. in vitro TNFα estimation
a. Protocol
b. Results
7. Inferences & Conclusion
8. References

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Immunomodulation
15/05/2007
Introduction
A global term that refers to a wide range of drug or
immunologic interventions that alter normal or abnormal
immune responses by
• Deletion of specific T or B cells
• Immune deviation
• Modification of various inflammatory pathways
(e.g., chemotaxis, adhesins, or intracytoplasmic
signaling).
Immunomodulators (agents that exhibit immunomodulatory
properties) can be categorized into
1. Immunosuppressants
2. Immunostimulants

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15/05/2007
Introduction
Silybum marianum
Silybum marianum (Blessed milk thistle) is a milk thistle
which belongs to the Asteraceae family which is a taxon of
dicotyledonous plants.
A fairly typical thistle with red and purple flowers and shiny
green leaves with white veins.
Originally, a native of Southern Europe through to Asia, it is
now found throughout the world.
The medicinal parts of the plant are the seeds and the
fruits.

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15/05/2007
Introduction
Silymarin (C25
H22
O10
)
A plant-derived flavonoid isolated from the seeds and fruits
of the Milk thistle, Silybum marianum.
Active principle was first isolated and characterized in
1968-1974 (Wagner et al, 1968)
Consists of 4 flavonolignan isomers (Wagner at al, 1974):
• Silybinin (70-80%)
• Isosilybinin (5%)
• Silydianine (10%)
• Silychristine (20%)

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15/05/2007
Silymarin
Properties
1. Hepatoprotective (liver protection)
2. Antidepressant (alleviate mental depression)
3. Antioxidant (maintains ROS levels)
4. Anti-inflammatory (inhibits PG, LTB synthesis; inhibits
Neutrophil migration)
5. Chemopreventive (anti-cancer properties)
6. Immunomodulator
7. Demulcent (soothes inflamed mucus membranes)
8. Laxative (stimulates bowel evacuation)
Introduction

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15/05/2007
Motivation
The work already done with Silymarin reveals to us its
multifarious utilities.
We know that the liver consists of Kuppffer cells
(specialized macrophages that form part of the RET
system). Since Silymarin is known to possess liver
restorative capacities, it can directly be linked to be
capable of exhibiting immune-stimulating activity.
This study focuses on determining the effect of
Silymarin on the Immune system and confirm
whether it does possess immune-stimulatory
activity or not.
Introduction
Motivation

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15/05/2007
Objectives of Work
• Study the immunomodulatory activity of
Silymarin (in vivo)
• Study the effect of Silymarin on stress-
induced mice (in vivo)
• Study the effect of Silymarin on intracellular
TNF-α secretion by isolated murine
neutrophils (in vitro)
Motivation
Objectives
of work
Introduction

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15/05/2007
Animals
Before Immunophenotyping
or Intracellular Cytokine
estimation can be performed,
the animals need to be
prepped.
Grouped into cages with 5/6
animals per cage and
marked with picric acid to
differentiate between the
mice.
Grouping
Head, Back, Tail
Head-Back, Back-Tail, No Color
Proper handling of mice
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Protocol for drug administration
Ø Challenge with antigen:
Challenging activates the immune
system.
Ø Day 0: Animals were challenged by
i.p. injection of SRBC (5 × 109
cells)
Ø Drug preparation: Using DW and
Gum Acacia
Ø Drug Admin: 2hrs after challenge.
For a period of 7 days.
Ø Dosage is measured as mg/kg
body wt. of the mice.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Protocol (contd.)
Ø Challenge with SRBC. Performed to assess primary
response.
Ø Cyclosporine (a known immunosuppressant) and
Levamisole (a known immunostimulant) used as
negative and positive controls respectively.
Ø 48hrs after challenge
(Day 8), blood drawn
from the ROP.
Ø Blood processed as
per the standardized
protocol and the sample
analyzed by Flow
Cytometry.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Study of Immunomodulatory activity
Immunomodulatory activity of Silymarin was studied by
using the following technique:
IMMUNOPHENOTYPING AND INTRACELLULAR
CYTOKINE ESTIMATION BY FLOW CYTOMETRY
Both IMMUNOPHENOTYPING and INTRACELLULAR
CYTOKINE ESTIMATION focus on the assessment of
lymphocyte populations in Acquired immunity.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Flow Cytometry
PRINCIPLE
• Uses principles of light scattering, light excitation and
emission of flourochrome molecules to generate
specific multi-parametric data from particles and cells
in the size range of 0.5µm to 40µm diameter.
• Lasers are most often used as a light source
WORKING
• Each suspended particle passing through the beam
scatters the light in some way. A combination of
scattered and fluorescent light is picked up by the
detectors and by analyzing fluctuations in brightness
at each detector (one for each fluorescent emission
peak) it is then possible to extrapolate various types of
information about the particles.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Immunophenotyping
A method that involves determining the cells of the immune
system by phenotyping them based on the cell surface
markers.
The surface markers analyzed in this study were:
1. CD8+ (T-cytotoxic cells)
2. CD4+ (T-helper cells)
To determine the presence of a particular type of cell, the
surface markers are tagged with flourochromes. When run
in a flow cytometer, the cells are separated and counted
based on the attached flourochrome.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Intracellular Cytokine Estimation
Cytokines : Signaling compounds secreted by Immune
cells, which help in regulating the activity of the immune
system by autocrine, paracrine and endocrine routes.
Estimation of the secreted cytokines (extracellular) is not
practically possible because they are not localized.
On the other hand, intracellular cytokines are localized
within the Golgi and hence can be targeted with
Flourochrome + MAbs directed toward the cytokines.
The cytokine analyzed in this study was IFNγ.
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
The flourochromes used for the experiments were:
1. Flourescein Iso Thio Cyanate (FITC)
2. Phycoerythrin (PE)
The flourochromes were attached to monoclonal
antibodies directed against the cell surface markers,
namely, CD4+, CD8+ and CD244 and the cytokines,
namely, IL12, IFNγ and TNFα
The combinations of flourochromes and MAbs used were:
• FITC – anti-CD4+
• PE – anti-CD8+
• FITC – anti-IFNγ
Flourochromes
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Results
Immunophenotyping
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Results
Intracellular Cytokine Estimation (IFNγ)
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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Stress
Stress
The constellation of events that begins with a stimulus
(stressor), which precipitates a reaction in the brain (stress
perception) that subsequently results in the activation of
certain physiologic systems in the body (stress response).
It refers to the condition where coping with the various
actual or perceived stimuli alters the homeostatic state of
the organism including behavioral, endocrine and
immunological changes.
2 types of stress are:
a. Acute Stress
b. Chronic Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Stress & the Immune System
All lymphocytes have adrenergic receptors. Differential
density and sensitivity of these receptors on lymphocytes
may affect responsiveness to stress
Sympathetic fibers release a wide variety of substances
that influence immune responses by binding to receptors
on white blood cells.
Acute stress generally has positive effect while chronic
stress typically provokes immunosuppression.
Psychological stress raises catecholamine levels, which
suppresses the immune system.
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Protocol
Ø Mice subjected to restraint stress in well-ventilated 50 ml
polypropylene tubes for 6 hours during the light cycle
during the experimental period of 12 days.
Ø After 6 hours of restraint, mice removed from tubes. 2
hours after de-stressing, drug is administered.
Ø After 12 days, blood drawn from ROP and processed as
per the standardized procedure and analyzed by flow
cytometry.
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction
Ø Groups:
Naïve control (not
subjected to any
stress);
Restraint stress
(RST) control;
RST + Drug

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15/05/2007
Results
Immunophenotyping (CD8+/CD4+)
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Immunophenotyping (CD244)
Results
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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Intracellular Cytokine Estimation (IFNγ, IL12)
Results
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Intracellular Cytokine Estimation (TNFα)
Results
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction

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15/05/2007
Motivation
Objectives
of work
Introduction
in vitro TNF-α secreted by
Neutrophils
Neutrophils are the most abundant type of WBCs and
are the first immune system cells to rush to the site of
an infection. Neutrophils are known as phagocytes
because they engulf foreign organisms and use
prepackaged chemicals to destroy them.
TNFα (Tumor Necrosis Factor) is a cytokine involved
in systemic inflammation and is a member of a group
of cytokines that stimulate the acute phase reaction.
On the in vitro activation of neutrophils by an agent,
TNFα is secreted.
The measure of the amount of TNFα secreted is a
good indicator of the level of activation of the immune
system.
Stress
Immunomodulatory
Study
in vitro TNFα

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15/05/2007
Motivation
Objectives
of work
Introduction
Protocol
Blood is drawn from naïve mice for this purpose. It is
processed and the neutrophils are isolated.
The isolated supernatant is incubated along with LPS
(Lipopolysaccharide - derived from E. coli) and the
drug at varying doses in a CO2
incubator.
A separate group called LPS control was taken, to
which no drug was added.
Following the 3 hr incubation, the flourochrome
tagged anti – TNFα MAb was added and the sample
was run in the flow cytometer to determine TNFα
levels.
Stress
Immunomodulatory
Study
in vitro TNFα

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15/05/2007
Motivation
Objectives
of work
Introduction
Results
Stress
Immunomodulatory
Study
in vitro TNFα

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15/05/2007
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction
Inferences & Conclusion
From the Immunophenotyping and Intracellular
Cytokine estimation data, Silymarin displayed
commendable dose dependent
immunopotentiating activity.
Most effective dose was 100 mg/kg body wt.
Its immune-potentiating activity was confirmed by
studying TNF-α secretion by activated Neutrophils in
vitro.
These studies actuated the test of its activity on mice
subjected to stress. The deleterious effect of chronic
stress was countered by Silymarin, which was able to
hype up the immune parameters to respectable
levels, thus demonstrating good stress-busting
activity.
Stress
in vitro TNFα
Conclusion

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15/05/2007
Stress
Immunomodulatory
Study
Motivation
Objectives
of work
Introduction
in vitro TNFα
References
References
Conclusion
Wagner H., Horhammer L., Munster R., 1968, The chemistry
of silymarin (silybin), the active principle of the fruits of
Silybum marianum (L.) Gaertn. (Carduus marianus) (L.).
Arzneimittelforschung, 18, 688-696
Wagner V.H., Diesel P., Seitz M., 1974, Chemistry and
analysis of silymarin from Silybum marianum (L.) Gaertn,
Arzneimittelforschung, 24, 466-471

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15/05/2007
Acknowledgements
Dr. K. Ramasamy
Dr. R. S. David Paul Raj
Dr. Sarang Bani
Ms. Beenish Khan
Ms. Prabhavathy Devan
Mr. Deepak Bhagwat
Ms. Anjali Pandey
Ms. Kiranjeet
Ms. Romaisa Latef
Thank you !!

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B. Tech Biotechnology Dissertation - SRM University - May 2007

B. Tech Biotechnology Dissertation - SRM University - May 2007

Vivek Krishnakumar

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