**4. Results**

## **4.1. Phytochemistry**

*Ginsenoside Compositon.* AQ extract contained a total ginsenoside content of 13.87% dry weight of extract and showed characteristics of *Panax quinquefolius* with Rb1 and Re as the predominant ginsenosides, with no detectable Rf and minimal levels of Rg1 (Fig.3).

*Crude PS extract.* The yield of crude PS fraction by four volumes of 95% ethanol precipitation was 10 % dry weight of root materials. A representative G-75 chromatographic profile of the crude PS extract is shown in Figure 4 [6]. The major PS peak (with a elution volume of 100 ML) had an estimated average molecular weight of 73kDa, while there were two minor, less well- resolved peaks (Fig 4 ).

*Monosaccharide composition of the WSPE determined by HPLC-ELSD.* Representative chromatograms showing individual monosaccharides in water soluble polysaccharide extract (WSPE) are shown in Figure 5 and 6. Glucose was found to be the major neutral monosaccharide present in WSPE with amounts ranging from 77 to 86 % (w/w). Galactose and arabinose were present in similar amounts with galactose being present at levels of 6.8 to 7.5 % (w/w) and arabinose being present at levels between 4.5 to 5.9 % (w/w). Galacturonic acid was also identified on WSPE at levels ranging from 8.7 to 9.5 % (w/w). Mannose and xylose were also monitored but were not detected in the sample.

**Figure 3.** HPLC ginsenoside profile of Ontario-grown American ginseng aqueous extract. A reversedphase Inspire C18 column was used with gradient elution consisted of [A] water and [B] acetonitrile: 0min, 80-20%; 0-60min, 58-42%; 60-70min, 10-90%; 70-80min, 80-20%. Absorbance of the eluates was monitored at 203nm.

**Figure 4.** Sephadex G-75 (47×2.5cm) chromatographic fractionation of PS extracts of ginseng. A calibrated column was loaded with 500mg of the extract, and then eluted with distilled water at a flow rate of 1mL/min. The y-axis is the absorbance at 230nm while the x-axis represents the elution volume (mL).

*Total fractionation of the DWSPE by ion exchange chromatography on DEAE-Cellulose.* To further analyse the WSPE, the material was de-proteinated using the Sevag method, giving a DWSPE with a yield of 92% and the protein content yield of 1.8% (relative to the dry weight of WSPE). Fractionation of WSPE by a combination of anion-exchange on DEAE-cellulose and gel permeation chromatographies with a procedure shown in Figure 1 revealed the elution of a neutral fraction with water (N-DWSPE, 75 % relative to the dry weight of DWSPE) and an acidic fraction (A-DWSPE, 4.5%) with a 0.5 M NaCl solution (Figure 7). Both fractions presented a wide and complex molecular weight distribution ranging from 5 to 410 KDa. The acidic fraction A-DWSPE containing most likely uronic acids was subjected to a second fractionation by DEAE-cellulose chromatography.

520 The Complex World of Polysaccharides

Rg1

Re

 **← PS →**

monitored at 203nm.

0 0.2 0.4 0.6 0.8 1 1.2 1.4

A U

0.00

0.02

0.04

0.06

0.08

(mL).

**ABSORBANCE AT 230N**

**M**

to 7.5 % (w/w) and arabinose being present at levels between 4.5 to 5.9 % (w/w). Galacturonic acid was also identified on WSPE at levels ranging from 8.7 to 9.5 % (w/w).

Rb1

**Figure 3.** HPLC ginsenoside profile of Ontario-grown American ginseng aqueous extract. A reversedphase Inspire C18 column was used with gradient elution consisted of [A] water and [B] acetonitrile: 0min, 80-20%; 0-60min, 58-42%; 60-70min, 10-90%; 70-80min, 80-20%. Absorbance of the eluates was

Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

**NORTH AMERICAN GINSENG POLYSACCHARIDE EXTRACT** 

ABS

Rc Rb2 Rd

**Figure 4.** Sephadex G-75 (47×2.5cm) chromatographic fractionation of PS extracts of ginseng. A calibrated column was loaded with 500mg of the extract, and then eluted with distilled water at a flow rate of 1mL/min. The y-axis is the absorbance at 230nm while the x-axis represents the elution volume

0 100 200 300 400 500 600 **ELUTION VOLUME (ML)**

*Total fractionation of the DWSPE by ion exchange chromatography on DEAE-Cellulose.* To further analyse the WSPE, the material was de-proteinated using the Sevag method, giving a

Mannose and xylose were also monitored but were not detected in the sample.

**Figure 5.** Representative chromatograms of the novel analytical method by HPLC-ELSD for the analysis of seven monosaccharides including glucose (Glu), galactose (Gal), arabinose (Ara), Xylose (Xyl), Mannose (Man) (A), galacturonic acid (Gal A) and rhamnose (Rha) (B).

**Figure 6.** Representative HPLC-ELSD Chromatograms of monosaccharide detected in WSPE, including glucose (Glu), galactose (Gal), arabinose (Ara) (A), galacturonic acid (Gal A) and rhamnose (Rha) (B).

*Secondary Fractionation with the A-DWSPE on DEAE-Cellulose column chromatography.* The elution of 0.6 g of A-DWSPE on a DEAE-cellulose chromatography was carried out using a stepwise gradient of NaCl. With these elution steps, A-DWSPE was separated into six fractions (Figure 2): A-DWSPE-1 (F1-F20, 96 mg, 16 %), A-DWSPE-2 (F21-F40, 54mg, 9%), A-DWSPE-3 (F41-F60, 162 mg, 27%), A-DWSPE-4 (F61-F80, 120mg, 20%), A-DWSPE-5 (81- F100, 78mg, 13%) and A-DWSPE-6 (F101-F120, 60mg, 10%) corresponding to the elution with 0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl solutions, respectively.

**Figure 7.** Representative chromatrograms by HPGPC-ELSD analysis; (**A**) DWSPE sample, (**B**) Dextran standards mixture, (**C**) DWSPE-N and (**D**) DWSPE-A. All samples were eluted under the same conditions.

In order to further analyze the unique NA ginseng polysaccharide samples, GPC with multiple detectors was utilized. Figure 8 shows the results of multiple pullulan polysaccharide standards ranging from 1800 Da to 1,050,000 Da. The utilized column gave good resolution of the individual standards over this range. Figure 8 shows the results of the multi-detector system containing 4 different detectors, i.e. refractive index (RI), right angle light scattering, low angle light scattering and viscometer. In Figure 9, the crude polysacchride extract is observed to have three major peaks with Mw values of 1092 kDa, 135 kDa and 12 kDa (Table 1). The major peak at 1092 kDa accounts for 66% of the weight fraction. Figure 9 shows the deproteinated polysaccharide fraction, in which the Mw values are largely unchanged (Table 1). However, the protein content fraction (PTF) contains only a small amount of carbohydrates with the majority of the high molecular weight fraction removed. The acid fraction contains 3 peaks, of generally lower Mw values, while the neutral fraction obtained from anionic exchange of DWSPE contains only one broad peak (Figure 9).

522 The Complex World of Polysaccharides

fractions (Figure 2): A-DWSPE-1 (F1-F20, 96 mg, 16 %), A-DWSPE-2 (F21-F40, 54mg, 9%), A-DWSPE-3 (F41-F60, 162 mg, 27%), A-DWSPE-4 (F61-F80, 120mg, 20%), A-DWSPE-5 (81- F100, 78mg, 13%) and A-DWSPE-6 (F101-F120, 60mg, 10%) corresponding to the elution

**Figure 7.** Representative chromatrograms by HPGPC-ELSD analysis; (**A**) DWSPE sample, (**B**) Dextran standards mixture, (**C**) DWSPE-N and (**D**) DWSPE-A. All samples were eluted under the same conditions.

In order to further analyze the unique NA ginseng polysaccharide samples, GPC with multiple detectors was utilized. Figure 8 shows the results of multiple pullulan polysaccharide standards ranging from 1800 Da to 1,050,000 Da. The utilized column gave

with 0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl solutions, respectively.

**Figure 8.** a) Resolution of individual pullulan standards: peak 1, 1800; peak 2, 5900; peak 3, 11,100; peak 4, 21,100; peak 5, 47,000; peak 6, 107,000; peak 8, 375,000; peak 9, 708,000; peak 10, 1,050,000 and b) calibration curve of pullulan standards through G-4000PWXL column.

**Figure 9.** a) GPC traces showing traces from the individual detectors of the PCE (Crude extract of polysaccharides), b) RI plot for the PCE (red), DWSPE (green) and PTF (black) samples. The high

molecular weight peak at retention volume 5.79mL has very low intensity in the PTF sample, which shows that this sample has a very low content of polysaccharide., c) RI plot of the neutral extract (black) and the acidic extract (green) from the ion exchange column, the neutral extract shows much bigger RI area, therefore much higher sample recovery.


**Table 1.** Molecular weight data of the PCE Extract.
