Separation and Characterization of Immuno-modulatory Polysaccharides of North American Ginseng Root (Panax quinquefolius L.)

Chike Azike

Department of Physiology and PharmacologyUniversity of Western Ontario

Outline of Presentation

• Background

• Rationale • Hypothesis

• Specific Aims • Experimental Approach

• Preliminary Results and Summary

• Significance and Conclusion

• 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• North American Ginseng (Panax quinquefolius) and Asian Ginseng (Panax ginseng).

Ginseng herb contains multiple bioactive compounds.

North American Ginseng root contains 2 types of bioactive compounds.

Ginseng

>30 Ginsenosides Polysaccharides (Poorly characterised ) May be >100Polysaccharides: complex chains of monosaccharides e.g. Glucose-Galactose-Mannose-Rabinose-Fucose...

Yuan Chun-su. 1993, Biochemical Pharmacology 58: 1685-1693

Sapogenin Steroidal Structure

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-saccharidespoly- 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 Acquired immunity

T-Lymphocyte B-Lymphocyte

DESTRUCTION OF PATHOGENS

Macrophage, Neutrophil, Dendritic Cell, Natural Killer Cell

inflammatory mediators (Cytokines, Reactive Intermediates)

Kelly Summers, 2007 Microbiology & Immunology

www.itb.cnr.it/.../UK/IDPagina/86/UT/systemPrint

Pathogen Associated Molecular Patterns (e.g. Lipopolysaccharide)

Up-regulation of inflammatory mediators (Cytokines & Reactive Oxygen / Nitrogen Species)

Macrophage Activation by Pathogen

Activation of Signalling Pathways

Activation of Kinases and Induction of Transcription factors (e.g NF-kB) and Gene expression

Mark Quinn. 2006, International Immunopharmacology 6: 317-313.

Difference in production profile of inflammatory mediators (Cytokines & Reactive Oxygen / Nitrogen Species)

Difference in Activation of Signalling Pathways

Difference in Activation of Kinases and Induction of Transcription factors (e.g NF-kB) and Gene expression

Multiple Receptors for Polysaccharides and Its Implication

?

?

Mark Quinn. 2006, International Immunopharmacology 6: 317-313.

Heterogeneous in nature

Rationale

Size difference Charge difference

Large medium Small Positive Negative + -

Ginseng Polysaccharides

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.

1. To isolate and fractionate crude polysaccharides into sub-fractions according to their molecular size and charge (ionization).

2a. By Chemical means.

3. To correlate between the chemical and immuno-modulatory properties among these polysaccharide sub-fractions.

Specific Aims

2b. By Pharmacological means ( the immuno-modulation)

2. Characterize sub-fractions of polysaccharides

Specific Aim 1:Isolate and fractionate crude polysaccharides into sub-fractions according to their molecular size and charge (ionization).

Arnason J.T, Challenges in Phytochemical analysis of Ginseng Components, Joint Conference of CICMR & OGIRC, Ontario 2008.

Polysaccharides

Supporting Cellulose Matrix

Experimental ApproachA. Isolate crude polysaccharides from Ginseng root powder. (Ginseng polysaccharides are water-soluble but have limited solubility in alcohol)

Supernatant Crude Polysaccharides

Aqueous extract (H2O soluble polysaccharides)

Precipitate with Ethanol(limited solubility in ethanol)

Ginseng root powder

Supernatant

Extract with ethanol

ResidueExtract with Hot H20

Residue

Ginsenosides(lipid soluble)

B. Fractionate crude polysaccharides according to their molecular size and charge (ionization) sequentially

Crude Polysaccharides

1. Size-Exclusion Chromatography according to difference in size

2. Ion-Exchange Chromatography according to difference in charge

Acidic Neutral Basic

Large Medium Small

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

Monosaccharides

Gas Chromatography Analysis

Acid Hydrolysis (H2SO4)

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.

Macrophage (RAW 264.7 Cells)

Stimulation of Inhibition of Inflammatory Mediators Inflammatory Mediators (Different polysaccharides induce different Inflammatory Mediators profile ?)

Treatment with polysaccharide sub-fractions

End points:

Up-regulation / Down-regulation of pro-inflammatory mediators such as:

Nitric Oxide (NO) Prostaglandin E2 (PGE2) Interleukin (IL-1, IL-6) Tumour Necrosis Factor alpha (TNF-α)

Preliminary Result

G-75 Frationation of Ginseng polysaccharide at 280nm

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Series1

G-25 FRACTIONATION OF GINSENG POLYSACCHARIDE

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•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.

The immuno-modulatory activity of North American Ginseng Extracts.

Ginsneng Crude Polysaccharide Extract Induced Nitrite Production in RAW 264.7 Murine Macrophages

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(uM) Nitrite Production

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 concentrated-response 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

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Cold-FX Polysaccharide

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Comparison of Inhibiton of LPS-Stimulated Effect of Ginseng Polysaccharide Extracts with Ginseng Aqueous and Ethanol Extracts & Cold-FX

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Cold-FX Polysaccharide

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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