**3.3. Pharmacological evaluation**

### *3.3.1. In vivo study*

Adult male rats (250-300 gm) were treated with 125 mg/kg of AQ extract or crude PS fraction dissolved in saline by gastric garvage (10 ml/Kg bwt) once daily for 3 or 6 consecutive days, and examined 24 hr after the last dose. Animals were anesthetized with i.m. injection (80 and 5 mg/kg b.wt. ketamine and xylazine, respectively) and the trachea was cannulated for lung bronchoalveolar lavage (BAL) with Dulbecco's phosphate-buffered saline (PBS) to collect alveolar macrophages.

Blood was collected into heparinized tubes from rats by intracardiac puncture, samples were immediately centrifuged at 3000 rpm for 10 minutes and the plasma was separated, aliquoted and stored at -20 °C until use.

#### *3.3.2. In vitro study*

#### *Cell culture*

Rat alveolar macrophages were collected by BAL using cannulated 10-ml syringe with three 10ml washes of PBS. Fluid recovered from BAL was centrifuged at 1000 rpm for 5 minutes. Cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 25mM HEPES, 2mM Glutamine, 100IU/ml penicillin and 100μg/ml streptomycin in 96-well tissue culture plates, at a density of 2×105 cells per well at 37°C maintained in a humidified incubator with 5% CO2.

#### *Cell treatment*

#### *Immuno-stimulatory effect*

Experiments to evaluate dose-related stimulation of inflammatory mediators profile *in vitro* were carried out by treating and incubating rat alveolar macrophages with 0, 50, 100 and 200μg/ml of ginseng extracts for 24 hours and washed before challenging with LPS (1μg/mL) was used as positive control. The 24 hours-production of NO, TNF-α and IL-6 in culture medium was determined.

#### *LPS-induced immuno-suppression*

518 The Complex World of Polysaccharides

ELSD temperature was set to 44 °C.

**3.3. Pharmacological evaluation** 

saline (PBS) to collect alveolar macrophages.

aliquoted and stored at -20 °C until use.

humidified incubator with 5% CO2.

culture medium was determined.

*3.3.1. In vivo study* 

*3.3.2. In vitro study* 

*Cell culture* 

*Cell treatment* 

*Immuno-stimulatory effect* 

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

Adult male rats (250-300 gm) were treated with 125 mg/kg of AQ extract or crude PS fraction dissolved in saline by gastric garvage (10 ml/Kg bwt) once daily for 3 or 6 consecutive days, and examined 24 hr after the last dose. Animals were anesthetized with i.m. injection (80 and 5 mg/kg b.wt. ketamine and xylazine, respectively) and the trachea was cannulated for lung bronchoalveolar lavage (BAL) with Dulbecco's phosphate-buffered

Blood was collected into heparinized tubes from rats by intracardiac puncture, samples were immediately centrifuged at 3000 rpm for 10 minutes and the plasma was separated,

Rat alveolar macrophages were collected by BAL using cannulated 10-ml syringe with three 10ml washes of PBS. Fluid recovered from BAL was centrifuged at 1000 rpm for 5 minutes. Cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 25mM HEPES, 2mM Glutamine, 100IU/ml penicillin and 100μg/ml streptomycin in 96-well tissue culture plates, at a density of 2×105 cells per well at 37°C maintained in a

Experiments to evaluate dose-related stimulation of inflammatory mediators profile *in vitro* were carried out by treating and incubating rat alveolar macrophages with 0, 50, 100 and 200μg/ml of ginseng extracts for 24 hours and washed before challenging with LPS (1μg/mL) was used as positive control. The 24 hours-production of NO, TNF-α and IL-6 in To examine the direct inhibitory effect of ginseng extracts on LPS-stimulated immune function, we pre-treated the macrophages with 0, 50, 100 or 200μg/ml of ginseng extracts for 24 hours and washed before challenging with LPS (1 μg/ml). The 24-hour cytokine production induced by LPS was determined by measuring NO, TNF-α and IL-6 levels in the culture medium.
