**5. Immunomodulatory activity**

524 The Complex World of Polysaccharides

**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

*In vivo effect*. Treatment with AQ extract for 3-6 days produced marked stimulation of alveolar macrophages as determined by increased production of NO and TNF- and IL-6 following culturing for 24 hrs, reaching activities that were 50-100% of the positive (LPS) control (Figure 10). This immunostimulatory effect was also reflected in the elevation of plasma TNF- and IL-6 levels of treated animals (Figure 11). However, the responsiveness of macrophages collected from ginseng treated animals to LPS stimulation *ex vivo* showed >50% to 100% reduction in NO, TNF-α and IL-6 production as compared to those non-ginseng treated controls, especially with 6 days of ginseng treatment (Figure 10). These data showed that orally administered AQ extract had both immuno-stimulatory and anti-inflammatory effect. Data presented in Figure 12 showed that this immunomodulatory activity could be extended to the PS extract on the basis of its effect on macrophage NO production and the LPS responsiveness.

**Figure 10.** Figure 10. Orally administered ginseng AQ extract: elevated cytokine production and reduced LPS-stimulatd cytokine production in cultured alveolar macrophages. Alveolar macrophages of rats treated orally with 0 and 125mg/kg ginseng AQ extract for 3 and 6 Days were cultured for 24 hours to measure production of NO and cytokines (by ELISA) . To determine responsiveness to

LPS stimulation, ginseng treated macrophages were exposed to 1ug/ml LPS in culture to determine changes in 24 hr NO and cytokine production . Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P<0.05 compared to the untreated control were statistically significant. Φ values in bracket denote fold increase in LPS-stimulated cytokine production over control.

**Figure 11.** Ginseng AQ extract treatment elevated plasma (i) TNF-α and (ii) IL-6 levels. Rats were treated orally with 125mg/kg ginseng AQ extract for 3 and 6 days . Plasma cytokine concentrations were determined by ELISA. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values *P*<0.05 compared to the untreated control were statistically significant.

**Figure 12.** Orally administered ginseng PS extract (125 mg/kg) up-regulated NO production and reduced responsiveness to LPS (LPS 1 μg/ml) stimulation in cultured alveolar macrophages. Cells from untreated controls were treated with LPS 1 μg/ml as positive control for macrophage responsiveness. NO was determined by Griess reaction assay. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P<0.05 compared to the untreated control were statistically significant. Φ values in bracket denote fold increase in LPS-stimulated NO production over control.

Bioactive Polysaccharides of American Ginseng *Panax quinquefolius* L. in Modulation of Immune Function: Phytochemical and Pharmacological Characterization 527

**80**

 **(i) Aqueous (AQ) extract** 

526 The Complex World of Polysaccharides

in LPS-stimulated cytokine production over control.

untreated control were statistically significant.

 **Control Ginseng Treatment**

**0**

 **Untreated**

**500**

**Plasma TNF-**

 **Production (pg/mL)**

**1000**

**1500**

**0**

**20**

**40**

**Nitrite Production (uM)**

increase in LPS-stimulated NO production over control.

**60**

**80**

**3 Days 6 Days**

\* \*

LPS stimulation, ginseng treated macrophages were exposed to 1ug/ml LPS in culture to determine changes in 24 hr NO and cytokine production . Three independent experiments were performed

**(i) TNF-α (ii) IL-6**

**Figure 11.** Ginseng AQ extract treatment elevated plasma (i) TNF-α and (ii) IL-6 levels. Rats were treated orally with 125mg/kg ginseng AQ extract for 3 and 6 days . Plasma cytokine

concentrations were determined by ELISA. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values *P*<0.05 compared to the

**(9.5) (5.3)**

**Ginseng Treatment**

**(1)**

**3 Days 6 Days**

\*

\*

 **Control Ginseng Treatment**

**\***

**0**

 **Untreated**

**200**

**400**

**Plasma IL-6 Production (pg/mL)**

**600**

**800**

 **Control 3 days 6 days**

**\***

**Figure 12.** Orally administered ginseng PS extract (125 mg/kg) up-regulated NO production and reduced responsiveness to LPS (LPS 1 μg/ml) stimulation in cultured alveolar macrophages. Cells from untreated controls were treated with LPS 1 μg/ml as positive control for macrophage responsiveness. NO was determined by Griess reaction assay. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P<0.05 compared to the untreated control were statistically significant. Φ values in bracket denote fold

compared to the untreated control were statistically significant. Φ values in bracket denote fold increase

and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P<0.05

 **(a) NO (b) TNF-α**

**Figure 13.** Immuno-stimulatory effects *in vitro* of AQ, crude PS, acidic PS and neutral PS extracts on 24 hours macrophage production of (a) NO and (b) TNF-α. Alveolar macrophages isolated from control rats were treated with 0, 50, 100 and 200μg/ml of ginseng extracts for 24 hours, and the culture supernatants were analysed for NO and TNF-α by Griess reaction assay and ELISA, respectively. Cells treated with LPS (1 μg/ml) were used as positive controls. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P < 0.05 compared to the untreated (vehicle) control were statistically significant.

#### *In vitro effect*

Both AQ, crude PS and acidic PS showed stimulation of NO and TNF-α production by alveolar macrophages *in vitro* (Figure 13). The magnitude of the response to PS was greater than those induced by acidic PS or AQ extract. Neutral PS was devoid of activity. The lack of

concentration-dependent effect of PS was probably due to its high potency and inducing its maximum effect at the concentration studied. Data presented in Figure 14 showed how pretreatment with various extracts for 24 hrs altered the subsequent response to LPS challenge. Since prior LPS treatment was known to cause desensitization of subsequent response to LPS, this was used as a positive control to evaluate the immunosuppressive effect of ginseng extracts. It was apparent that PS was the most effective in reducing the NO and TNF-α response to LPS, while the AQ and acidic PS extracts were similar, and neutral PS was inactive.

> > **LPS**

**LPS-LPS**

 **(a) NO (b) TNF-α**

**(ii) Crude PS extract** 

**50**

**Treatment Concentration (ug/mL)**

**100**

**200**

**(iii) Acidic PS Extract** 

**LPS-LPS**

\*

**50**

**Treatment Concentration (ug/mL)**

\*

**100**

\*

**200**

\*

**LPS**

**Nitrite Production (uM)**

**Figure 14.** *In vitro* effects of AQ, crude PS, acidic PS and neutral PS extracts on LPS-stimulated 24 hours macrophage production of (a) NO and (b) TNF-α. Rat Alveolar macrophages were pre-treated with

ginseng extracts (0, 50, 200 μg/ml) for 24 hours and were washed before challenged with LPS 1 μg/ml. 24 hr-pretreatment with LPS prior to LPS stimulation was used as positive control to demonstrate desensitization of macrophage responsiveness (LPS-LPS). NO and TNF-α were determined by Griess reaction assay and ELISA, respectively. Three independent experiments were performed and the data were shown as mean ± SD. Datasets were evaluated by ANOVA. \* Values P < 0.05 compared to the LPS positive control were statistically significant.
