Transcript Separation and Characterization of Immuno

Separation and Characterization of Immunomodulatory Polysaccharides of North American
Ginseng Root (Panax quinquefolius L.)
Chike Azike
Department of Physiology and Pharmacology
University of Western Ontario
Outline of Presentation
• Background
• Rationale
• Hypothesis
• Specific Aims
• Experimental Approach
• Preliminary Results and Summary
• Significance and Conclusion
Ginseng
• North American Ginseng (Panax quinquefolius)
and Asian Ginseng (Panax ginseng).
• Member of the Araliaceae family of plants.
Indigenous to North America with over 400 years historical
use. Cultivated for its highly valued root which have
depleted over the years in the wild due to over utilisation.
• Traditionally Ginseng is used as an Adaptogen and Tonic;
to reduce stress, improve resistance to disease and
restore health.
• Few studies have been done on North American
Ginseng.
• Canada is the largest producer of North American Ginseng
2.5 million Kg worth of $83 million exported in 2007.
Ginseng herb contains multiple bioactive compounds.
North American Ginseng root contains 2 types of bioactive
compounds.
Ginseng
>30 Ginsenosides
Sapogenin Steroidal Structure
Polysaccharides
(Poorly characterised )
May be >100
Polysaccharides:
complex chains of monosaccharides
e.g. Glucose-Galactose-MannoseRabinose-Fucose...
Yuan Chun-su. 1993, Biochemical Pharmacology 58: 1685-1693
http://fshn.ifas.ufl.edu/faculty/mrmarshall/fos4311/presentations/Polysaccharides
• Reports from Modern day Scientific Research suggests Ginseng
Polysaccharides possess the following pharmacological
activities:
-Anti-tumor
-Radioprotection
-Hypoglycemia
-Regulation of Immune system
-Activation of hepatic function
-Anti-stress
-Anti-fatigue
-Anti-inflammatory
-Anti-ulcer
-Enhancement of brain function
-Antioxidant
-Neuroprotection
-Cardioprotection
Yuan Chun-su. 1993, Biochemical Pharmacology 58: 1685-693
Interest in Studying Ginseng Polysaccharides?
Most plant-derived polysaccharides display:
• Variety of pharmacological activities.
• Play key role in cell-cell signalling, have been recognized as
new target for drug discovery.
• Biological response modifier which regulates immune system.
• Ginseng Polysaccharides
Cold-FX preparation, extract of Ginseng root containing
poly- furanosyl- pyranosyl-saccharides is used clinically
for the prevention and treatment of common cold.
Mark Quinn. 2006, International Immunopharmacology 6: 317-313.
IMMUNE SYSTEM
Innate immunity
Macrophage,
Neutrophil, Dendritic
Cell, Natural Killer Cell
Acquired immunity
T-Lymphocyte B-Lymphocyte
inflammatory mediators (Cytokines, Reactive Intermediates)
DESTRUCTION OF PATHOGENS
Kelly Summers, 2007 Microbiology & Immunology
www.itb.cnr.it/.../UK/IDPagina/86/UT/systemPrint
Macrophage Activation by Pathogen
Pathogen Associated Molecular
Patterns (e.g. Lipopolysaccharide)
Activation of Signalling Pathways
Activation of Kinases
and
Induction of Transcription factors
(e.g NF-kB) and Gene expression
Up-regulation of inflammatory mediators
(Cytokines & Reactive Oxygen / Nitrogen Species)
Mark Quinn. 2006, International Immunopharmacology 6: 317-313.
Multiple Receptors for Polysaccharides
and Its Implication
Difference in Activation of Signalling Pathways
?
Difference in Activation of Kinases
and
Induction of Transcription factors
(e.g NF-kB) and Gene expression
?
Difference in production profile of inflammatory mediators
(Cytokines & Reactive Oxygen / Nitrogen Species)
Mark Quinn. 2006, International Immunopharmacology 6: 317-313.
Rationale
Ginseng Polysaccharides
Heterogeneous in nature
Charge difference
Size difference
Large
medium
Small
Positive
+
Negative
-
Expect to induce different profile of Inflammatory
mediators and Immuno-modulating activities
Hypothesis
Polysaccharides of North American
Ginseng root can be fractionated into
multiple components according to their
physico-chemical characteristics
(molecular size and charge) and these
components possess different
immuno-modulating activities.
Specific Aims
1. To isolate and fractionate crude polysaccharides into
sub-fractions according to their molecular size
and charge (ionization).
2. Characterize sub-fractions of polysaccharides
2a. By Chemical means.
2b. By Pharmacological means
( the immuno-modulation)
3. To correlate between the chemical and immuno-modulatory
properties among these polysaccharide sub-fractions.
Specific Aim 1:
Isolate and fractionate crude polysaccharides
into sub-fractions according to their molecular
size and charge (ionization).
Polysaccharides
Supporting Cellulose Matrix
Arnason J.T, Challenges in Phytochemical analysis of Ginseng
Components, Joint Conference of CICMR & OGIRC, Ontario 2008.
Experimental Approach
A. Isolate crude polysaccharides from Ginseng root powder.
(Ginseng polysaccharides are water-soluble but have
limited solubility in alcohol)
Ginseng root powder
Extract with ethanol
Supernatant
Ginsenosides
(lipid soluble)
Residue
Extract with Hot H20
Aqueous extract (H2O soluble polysaccharides) Residue
Supernatant
Precipitate with Ethanol
(limited solubility in ethanol)
Crude Polysaccharides
B. Fractionate crude polysaccharides according to their
molecular size and charge (ionization) sequentially
Crude Polysaccharides
1. Size-Exclusion Chromatography
according to difference in size
Large
Medium
Small
2. Ion-Exchange Chromatography
according to difference in charge
Acidic
Neutral
Basic
http://www.chem.iitkgp.ernet.in/faculty/SDG/Protein%20isolation%20chromatography.pdf
Specific Aim 2a:
Characterisation by Chemical means:
Characterize composition of the polysaccharide
sub-fractions by chemical means.
A. Monosaccharide content.
Polysaccharide sub-fractions
Acid Hydrolysis (H2SO4)
Monosaccharides
Gas Chromatography Analysis
B. Measure concentration, molecular weight and conformation
(branched or linear nature) of polysaccharides by
Gel Permeation Chromatography using different detectors:
Refractive Index Detector
provides information on concentration of polysaccharides.
Light Scattering Detector
provides information on molecular weight of polysaccharides.
Viscometer Detector
provides information on conformation (branched or linear) of
polysaccharides.
Specific Aim 2b.
Characterisation by Pharmacological means
Characterize the immuno-modulatory properties of
these polysaccharide sub-fractions.
Treatment with polysaccharide sub-fractions
Macrophage (RAW 264.7 Cells)
Stimulation of
Inhibition of
Inflammatory Mediators
Inflammatory Mediators
(Different polysaccharides
induce different Inflammatory
Mediators profile ?)
End points:
Up-regulation / Down-regulation of proinflammatory mediators such as:
Nitric Oxide (NO)
Prostaglandin E2 (PGE2)
Interleukin (IL-1, IL-6)
Tumour Necrosis Factor alpha (TNF-α)
Preliminary Result
•G-75 Sephadex Column (97x2.5cm) and G-25 Sephadex (30x2.5cm) Chromatography of
Ginseng Polysaccharide Extract resulted in the fractionation of one peak.
Suggesting the molecular weight of the Ginseng polysaccharides to be between 25,000 to 75,000.
Absorbance at 280nm
G-75 Frationation of Ginseng polysaccharide at
280nm
0.6
0.5
0.4
0.3
Series1
0.2
0.1
0
0
100
200
300
400
500
600
Elution Volume (mL)
ABSORBANCE AT
230NM
G-25 FRACTIONATION OF GINSENG
POLYSACCHARIDE
1
0.8
0.6
ABS
0.4
0.2
0
0
100
200
300
VOLUME (ML)
400
500
The immuno-modulatory activity of North American
Ginseng Extracts.
60
50
40
30
20
10
0
5000
2000
1000
500
200
100
50
10
Nitrite Production
Untreated
Control
Medium
Nitrite Concentration
(uM)
Ginsneng Crude Polysaccharide Extract Induced Nitrite Production in RAW 264.7
Murine Macrophages
Concentration (ug/mL)
The production of Nitric Oxide (NO) was measured indirectly as a function of
the nitrite concentration in the culture medium.
Crude Polysaccharide (PS) extract of North American Ginseng
root induced the production of nitrite in a concentratedresponse manner from 10 to 500ug/mL, with maximum effect at
observed at 500ug/mL. While high concentrations from
1000ug/mL to 5000ug/mL exhibited reduction in the production
of nitrite.
The reduction in nitrite production at higher concentrations was
further examined by cell viability assay to verify if the
decreased effect was as a result of cell death of the
macrophages.
This Cell Viability (MTT) assay showed that Crude
Polysaccharide extract of North American Ginseng root was not
cytotoxic at 10-500ug/mL, but at higher concentrations of
1000ug/mL to 5000 ug/mL cell death occurred which was
responsible for the observed reduction in nitrite production.
Comparison of the Immunomodulatory effects
(Immuno-stimulation and Inhibition of LPS-Stimulation) of Ginseng
Polysaccharide Extracts with Aqueous and Ethanolic extracts
Comparison of the Immunostimulatory Effects of Ginsneng Polysaccharide Extracts with Ginseng
Aqueous and Ethanol Extracts & Cold-FX
LPS
Positive
Control
70
Fractionated
Polysaccharide
Crude
Polysaccharide
Aqueous
Extract
60
50
Cold-FX Polysaccharide
40
Immunostimulation
30
Ethanol
Extract
20
10
Concentration (ug/mL)
50
10
0
20
0
50
0
50
10
0
20
0
50
0
50
10
0
20
0
50
0
50
10
0
20
0
50
0
50
10
0
20
0
50
0
S1
LP
ate
d
0
Un
tre
Nitrite concentration (uM)
80
100
LPS
Positive
Control
80
Fractionated
Polysaccharide
Crude
Polysaccharide
Aqueous
Extract
Cold-FX
Polysaccharide
60
Ethanol
Extract
Inbition of LPS-Stimulation
40
20
Concentration (ug/mL)
200
50
500
100
200
50
500
100
200
50
LPS-1
0
Untreated
Nitrite Concentration (uM)
Comparison of Inhibiton of LPS-Stimulated Effect of Ginseng
Polysaccharide Extracts with Ginseng Aqueous and Ethanol Extracts & Cold-FX
Summary
1. The Polysaccharide extracts behave like the Aqueous extract in that it
demonstrated pro-inflammatory (immunostimulatory) activity by up-regulating NO
production with potency similar to that of Cold-FX in a dose-response relationship.
This apparent immunostimulatory effect is supportive of the traditional herbal
medicine use of ginseng as an adaptogen to counteract various forms of stress
including that of the immune system caused by microbial pathogens. This suggests
that the polysaccharides contribute to immunostimulatory effects observed in
aqueous extracts of North American Ginseng root.
2. The polysaccharide extracts has no anti-inflammatory effect in that it failed to
inhibit lipopolysaccharide (LPS)-stimulated NO production unlike the ethanol extract,
when incubated 2 hours prior to the addition of LPS. This suggests that ginsenosides
present in the ethanol extracts are responsible for the anti-inflammatory effects of
North American ginseng root.
3. The observed difference in the pharmacological activity due to differences in the
photochemical bioactive components of North American Ginseng root suggests that
the polysaccharides and aqueous extracts will be useful for infected and stressed
immune systems while the alcoholic extracts will have good application in chronic
inflammatory diseases.
Next Plan of Action:
Fractionate Ginseng Polysaccharides by Ion-Exchange
Chromatography according to difference in charge.
1. Characterize composition of the polysaccharide sub-fractions by
chemical means to obtain;
a. Monosaccharide content
b. Absolute molecular weight
c. Conformation (branched or linear nature) of polysaccharides by
Gel Permeation Chromatography using different detectors
2. Identify potent immuno-modulatory polysaccharide fractions and
relate their immuno-stimulatory or inhibitory properties to their
chemical properties such as molecular weight, branching
(conformation) and charge.
Significance and Conclusion
Results obtained from this study will be useful
in developing more potent and specific
immuno-stimulatory and immuno-inhibitory
Ginseng polysaccharide products.
Acknowledgement
Dr. Ed Lui
Dr. Paul Charpentier
Dr. Dave Freeman
Dr. Jirui Hou
Mrs. Hua Pei.
Members of Dr. Ed Lui’s Research Group
Funding: Ontario Research Fund Excellence Program,
Ministry of Research and Innovation