Separation and Characterization of Immuno-modulatory 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 Polysaccharides (Poorly characterised ) May be >100 Polysaccharides: complex chains of monosaccharides e.g. Glucose-Galactose-Mannose-Rabinose-Fucose... Sapogenin Steroidal Structure 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.

Innate immunity Acquired immunity IMMUNE SYSTEM Innate immunity Acquired immunity Macrophage, Neutrophil, Dendritic Cell, Natural Killer Cell 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 Size difference Charge 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 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.

Isolate and fractionate crude polysaccharides 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 Residue Ginsenosides (lipid soluble) Extract with Hot H20 Aqueous extract (H2O soluble polysaccharides) Residue Precipitate with Ethanol (limited solubility in ethanol) Supernatant 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 http://www.chem.iitkgp.ernet.in/faculty/SDG/Protein%20isolation%20chromatography.pdf

Characterisation by Chemical means: 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.

Characterisation by Pharmacological means 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 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 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. 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 LPS Positive Control Fractionated Polysaccharide Crude Polysaccharide Aqueous Extract Cold-FX Polysaccharide Ethanol Extract

LPS Positive Control Fractionated Polysaccharide Crude Polysaccharide Aqueous Extract Cold-FX Polysaccharide Ethanol Extract

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; Monosaccharide content Absolute molecular weight 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.

in developing more potent and specific 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.

Dr. Ed Lui Dr. Paul Charpentier Dr. Dave Freeman Dr. Jirui Hou 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