**3. Methods**

**Preparation of the aqueous (AQ) and polysaccharide (PS) ginseng extracts.** Dried ginseng root samples were shipped to Naturex (USA) for extraction. Samples were ground between ¼ and ½ inch and used to produce the AQ extract [6]. Briefly, 4kg ground ginseng roots were soaked three times during five hours in 16L of water solution at 40°C. After extraction, the solution was filtered at room temperature. The excess solvent was then removed by a rotary evaporator under vacuum at 45°C. The three pools were combined and concentrated again until the total solids on a dry basis were around 60%. These concentrates were lyophilized with a freeze dryer (Labconco, USA) at -50°C under reduced pressure to produce the AQ ginseng extract in powder form. Yield of the powder extracts from the concentrates was about 66%. The yields of the final extract (mean **±** standard deviation of % extractive) from the initial ground root were 41.74**±**4.92.

A solution of AQ extract in distilled water (10g/10mL) was prepared, and the crude PS was precipitated by the addition of four volumes of 95% ethanol. The PS fraction was collected by centrifugation at 350×*g* (Beckman Model TJ-6, USA) for 10 minutes and lyophilized to produce the crude PS extract.

To prepare the water soluble polysaccharide extract (WSPE), ginseng roots (500 g) were extracted with 7.0 L of MilliQ (EMD Millipore) water at 100 ºC for 4 h and filtered through sheets of glass fiber. The solid material was extracted twice under identical conditions. The filtrates were combined, then centrifuged to remove water insoluble materials and supernatants were concentrated (1.0 L) and precipitated by the addition of 95% ethanol (4 to 1 volumes). After centrifugation, the precipitate was washed and dried by solvent exchange, first using 95% ethanol, and then absolute ethanol. Crude water soluble polysaccharide extract (WSPE) was obtained with a 20.0 % yield (relative to dry weight of plant material) (Figure 1).

## **3.1. Chromatography of ginseng extracts**

#### *High performance liquid chromatography (HPLC) analysis for ginsenoside determination*

HPLC analysis on the composition of ginsenosides in the AQ extracts (100mg/ml methanol) was performed with a Waters 1525 HPLC System with a binary pump and UV detector [6]. A reversed-phase Inspire C18 column (100mm×4.6 mm, i.d. 5μm) purchased from Dikma Technologies (USA) was used for all chromatographic separations. Gradient elution consisted of [A] water and [B] acetonitrile at a flow of 1.3mL/min as follows: 0min, 80-20%; 0-60min, 58-42%; 60-70min, 10-90%; 70-80min, 80-20%. Absorbance of the eluates was monitored at 203nm.

#### *Sephadex G-75 chromatography*

514 The Complex World of Polysaccharides

separation as well as quantitative and qualitative analysis.

evaluate its immunomodulatory activity.

**2.1. Chemicals and biologicals** 

approved the study (Protocol No: 2009-070).

**2. Materials** 

**3. Methods** 

rarely studied in the North American species. They are made up of a complex chain of monosaccharides rich in L-arabinose, D-galactose, L-rhamnose, D-galacturonic acid, Dglucuronic acid and D-galactosyl residues [7]. The actual structural characteristics and the heterogeneity of PS components are poorly understood due to a lack of methodologies for

Most studies have focused on Asian ginseng PS and mostly *in vitro* experimental models. In this chapter, we will focus on *Panax quinquefolius* (American ginseng). The use of gel permeation chromatography (GPC) with multiple detectors has provided enhanced resolution for phytochemical analysis. And we have used both *in vivo* and *in vitro* models to

**Ginseng**. Four-year-old American ginseng roots collected in 2007 from five different farms in Ontario, Canada were provided by the Ontario Ginseng Growers Association. Ginseng extracts from each farm were prepared individually and combined to produce composite extracts which were used for phytochemical and pharmacological studies [6]. The AQ

Sephadex G75 was purchased from GE Healthcare Bio-Sciences AB (Sweden). The Diethylaminoethyl (DEAE)-Cellulose and monosaccharide standards were purchased from Sigma (Oakville, Ontario). All other chemicals were of analytical grade and used as received. Cell culture medium and reagents were purchased from Gibco laboratories (USA). BD OptEIA ELISA kits tumour necrosis factor-α and interleukin-6 (BD Biosciences, USA). LPS from *Escherichia coli* and Griess reagent were purchased from Sigma-Aldrich (USA).

*Animals*. Adult male Sprague-Dawley rats (200–250g; Charles River, St. Constant, QC, Canada) were used. The Animal Ethics Review Committee of the Western University

**Preparation of the aqueous (AQ) and polysaccharide (PS) ginseng extracts.** Dried ginseng root samples were shipped to Naturex (USA) for extraction. Samples were ground between ¼ and ½ inch and used to produce the AQ extract [6]. Briefly, 4kg ground ginseng roots were soaked three times during five hours in 16L of water solution at 40°C. After extraction, the solution was filtered at room temperature. The excess solvent was then removed by a rotary evaporator under vacuum at 45°C. The three pools were combined and concentrated again until the total solids on a dry basis were around 60%. These concentrates were lyophilized with a freeze dryer (Labconco, USA) at -50°C under reduced pressure to produce the AQ ginseng extract in powder form. Yield of the powder extracts from the

extract has no detectable endotoxin contamination as determined by Limulus test.

AQ ginseng extract (500mg) was dissolved in 5mL distilled water and then fractionated by loading to a calibrated Sephadex G-75 column (47×2.5cm) equilibrated and eluted with distilled water mobile phase at 4°C with a flow rate of 1mL/min [6]. Absorbance of the eluates was monitored at 230nm. Fractions were collected and lyophilized for the study of bioactivity distribution.

#### *Preparation of the de-proteinated water soluble polysaccharide extracts (DWSPE)*

WSPE (50 g) were re-dissolved in 1.5 L of MilliQ water and partitioned five times with Sevag reagent (1:4 n-butanol:chloroform, v/v, 500 mL each) to remove proteins [8]. Polysaccharides were precipitated again by ethanol and dried by solvent exchange. This procedure yielded 46.0 g of the de-proteinated water soluble polysaccharide fraction (DWSPE). The procedure for the preparation of DWSPE from *P. quinquefolius* is shown in Figure 1.

#### *Total fractionation of the DWSPE by ion exchange chromatography on DEAE-Cellulose*

DWSPE fraction (20 g) was dissolved in MilliQ water (200 mL) and loaded on a DEAE-Cellulose column (10.0 X 20 cm, Cl- ) pre-equilibrated with MilliQ water. The column was eluted first with 4.0 L of MilliQ water at a flow rate of 10 mL/min (4 bar column pressure) to obtain the unbound or neutral fraction (DWSPE-N) and then with 4.0 L of 0.5 M NaCl to obtain the bound or acidic fraction (DWSPE-A). The fractions were concentrated, dialyzed (cut off pore size of 2 KDa) against MilliQ water and freeze dried to give 15.0 g (13.8 %) of the DWSPE-N and 0.9 g (0.83 %) of the acidic fraction DWSPE-A. DWSPE-A (0.6 g) was dissolved in 50 mL MilliQ water and loaded on a DEAE-Cellulose column (10 X 20 cm, Cl- ). The column was eluted by a stepwise gradient with 2.0 L of NaCl aqueous solutions (0.0, 0.1, 0.2, 0.3 and 0.5 M each) at a flow rate of 10 mL/min (4 bar column pressure). A total of 120 eluate fractions were collected (50 mL each), dialyzed and lyophilized (Figure 2).

#### *High performance gel permeation chromatography-evaporative light scattering detection (HPGPC-ELSD) analysis*

HPGPC was carried out at 40 ºC using a TSK-gel G-3000PWXL column (7.8 X 300 mm, TOSOH, Japan) connected to a HPLC system coupled with Diode Array and Evaporative Light Scattering Detectors (DAD-ELSD). The column was calibrated with standard dextrans (5 to 410 KDa range, Figure 7-B). Ten microliters of 20 mg/mL solutions of DWSPE, DWSPE-N and DWSPE-A were separately injected and eluted with HPLC grade water at a flow rate of 0.8 mL/min and monitored using ELSD with a temperature setting at 80 °C.

#### *High performance gel permeation chromatography-multi-detector analysis*

AQ and PS ginseng extracts were analysed at 40°C with TSK-gel PWXL G-4000PWXL column (7.8 X 300 mm, TOSOH, USA) connected to a Viscotek (Varian Instruments, USA) gel permeation chromatography system with Omnisec software (version 4.5, Viscotek, USA) for data acquisition. Solutions of AQ and PS extract (1mg/mL) were filtered with 0.2μm nylon filter and used for analysis. Each sample (100μl) was injected and eluted with 0.3M sodium chloride (NaCl) mobile phase at a flow rate of 1mL/min and monitored using a multiple detectors system for light scattering, refractive index and viscosity. Pullulan polysaccharide reference standard was analyzed as a positive control.

#### **3.2. Analysis of the monosaccharides composition in WSPE by HPLC-ELSD**

Carbohydrate analysis represents a challenge in analytical chemistry since neutral or acidic saccharides (mono, oligo and poly) have little UV activity. In our study, evaporative light scattering detection (ELSD) was used. The ELSD does not require the solutes of interest to have any optical properties; and the only requirement is that the eluent be more volatile than the solutes. WSPE (20 mg) was dissolved in 10 mL of 2N HCl solution and was boiled for 2 h. The hydrolyzed product was neutralized (pH 6-7) and centrifuged before analysis.

Bioactive Polysaccharides of American Ginseng *Panax quinquefolius* L. in Modulation of Immune Function: Phytochemical and Pharmacological Characterization 517

516 The Complex World of Polysaccharides

*ELSD) analysis* 

centrifuged before analysis.

Cellulose column (10.0 X 20 cm, Cl-

*Total fractionation of the DWSPE by ion exchange chromatography on DEAE-Cellulose*

DWSPE fraction (20 g) was dissolved in MilliQ water (200 mL) and loaded on a DEAE-

eluted first with 4.0 L of MilliQ water at a flow rate of 10 mL/min (4 bar column pressure) to obtain the unbound or neutral fraction (DWSPE-N) and then with 4.0 L of 0.5 M NaCl to obtain the bound or acidic fraction (DWSPE-A). The fractions were concentrated, dialyzed (cut off pore size of 2 KDa) against MilliQ water and freeze dried to give 15.0 g (13.8 %) of the DWSPE-N and 0.9 g (0.83 %) of the acidic fraction DWSPE-A. DWSPE-A (0.6 g) was dissolved in 50 mL MilliQ water and loaded on a DEAE-Cellulose column (10 X 20 cm, Cl-

The column was eluted by a stepwise gradient with 2.0 L of NaCl aqueous solutions (0.0, 0.1, 0.2, 0.3 and 0.5 M each) at a flow rate of 10 mL/min (4 bar column pressure). A total of

*High performance gel permeation chromatography-evaporative light scattering detection (HPGPC-*

HPGPC was carried out at 40 ºC using a TSK-gel G-3000PWXL column (7.8 X 300 mm, TOSOH, Japan) connected to a HPLC system coupled with Diode Array and Evaporative Light Scattering Detectors (DAD-ELSD). The column was calibrated with standard dextrans (5 to 410 KDa range, Figure 7-B). Ten microliters of 20 mg/mL solutions of DWSPE, DWSPE-N and DWSPE-A were separately injected and eluted with HPLC grade water at a flow rate

AQ and PS ginseng extracts were analysed at 40°C with TSK-gel PWXL G-4000PWXL column (7.8 X 300 mm, TOSOH, USA) connected to a Viscotek (Varian Instruments, USA) gel permeation chromatography system with Omnisec software (version 4.5, Viscotek, USA) for data acquisition. Solutions of AQ and PS extract (1mg/mL) were filtered with 0.2μm nylon filter and used for analysis. Each sample (100μl) was injected and eluted with 0.3M sodium chloride (NaCl) mobile phase at a flow rate of 1mL/min and monitored using a multiple detectors system for light scattering, refractive index and viscosity. Pullulan

**3.2. Analysis of the monosaccharides composition in WSPE by HPLC-ELSD** 

Carbohydrate analysis represents a challenge in analytical chemistry since neutral or acidic saccharides (mono, oligo and poly) have little UV activity. In our study, evaporative light scattering detection (ELSD) was used. The ELSD does not require the solutes of interest to have any optical properties; and the only requirement is that the eluent be more volatile than the solutes. WSPE (20 mg) was dissolved in 10 mL of 2N HCl solution and was boiled for 2 h. The hydrolyzed product was neutralized (pH 6-7) and

120 eluate fractions were collected (50 mL each), dialyzed and lyophilized (Figure 2).

of 0.8 mL/min and monitored using ELSD with a temperature setting at 80 °C.

*High performance gel permeation chromatography-multi-detector analysis*

polysaccharide reference standard was analyzed as a positive control.

) pre-equilibrated with MilliQ water. The column was

).

**Figure 1.** Preparation of the water soluble polysaccharides extract (WSPE) and the de-proteinated extract from the roots of 4-year-old Ontario-grown Panax quinquefolius.

**Figure 2.** Step wise fractionation of the de-proteinated water soluble ginseng polysaccharide extract (DWSPE).

HPLC analysis was conducted using an 1100 series HPLC-DAD-ELSD system (Agilent Technologies Inc., Santa Clara, CA, USA). To enhance resolution of monosaccharides, analysis was performed using two separate columns (Figure 2). Glucose, galactose, arabinose, mannose and xylose were eluted using a Rezex RPM Monosaccharide PB+2 (8%) (Phenomenex, Torrance, California) column with a mobile phase of 100 % water (Chromasolv Plus, HPLC grade) isocratically at 80 °C and a flow rate of 0.6 mL/min. The ELSD temperature was set to 80 °C. Galacturonic acid and rhamnose content were examined using a Luna 5 μ NH2 100Å column (Phenomenex, Torrance, California) and eluted with a mobile phase of acetonitrile and water (80:20) at 40 °C and a flow rate of 3 mL/min. The ELSD temperature was set to 44 °C.
