**6. Discussion**

528 The Complex World of Polysaccharides

 **(i) AQ Extract** 

**LPS**

**(ii) Crude PS extract** 

**LPS**

**(iii) Acidic PS Extract** 

**LPS**

**(iv) Neutral PS Extract** 

**LPS**

**LPS-LPS**

\*

**LPS-LPS**

\*

**LPS-LPS**

\*

**Nitrite Production (uM)**

**Nitrite Production (uM)**

**Nitrite Production (uM)**

**Nitrite Production (uM)**

**LPS-LPS**

\*

**50**

**50**

**Treatment Concentration (ug/mL)**

**50**

**Treatment Concentration (ug/mL)**

**50**

**Treatment Concentration (ug/mL)**

**100**

**200**

\*

\*

**100**

\*

**200**

\*

**100**

\* \* \*

**Treatment Concentration (ug/mL)**

\*

**100**

\*

**200**

**200**

**LPS**

**LPS**

**LPS**

**LPS**

**TNF- Production (pg/mL)**

**LPS-LPS**

**LPS-LPS**

\*

**TNF Production (pg/mL)**

**LPS-LPS**

\*

**50**

**Treatment Concentration (ug/mL)**

**50**

**50**

**Treatment Concentration (ug/mL)**

**Treatment Concentration (ug/mL)**

\* \* \* \*

**100**

**100**

\* \* \*

**200**

**200**

**100**

\* \* \*

**200**

**TNF- Production (pg/mL)**

**LPS-LPS**

\*

**50**

**Treatment Concentration (ug/mL)**

\*

**100**

**200**

\* \*

\*

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,

**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

**TNF- Production (pg/mL)**

while the AQ and acidic PS extracts were similar, and neutral PS was inactive.

 **(a) NO (b) TNF-α**

Medicinal plants have been in use for human health for thousands of years, yet polysaccharides have only been recognized recently as a major contributor to the bioactivity of these traditional medicines. Polysaccharides from plant sources with immunomodulatory, anti-tumor, anti-viral, anti-bacterial, anti-inflammatory, anti-oxidant, and anti-diabetogenic activities have been reported [2, 9-15]. And a few polysaccharides, including lentinan, *Astragalus* polysaccharide, polyporus polysaccharide and *Achyranthes bidentata* polysaccharide have been licensed for clinical application in China [16]. In Canada, a polysaccharide-enriched American ginseng extract (Cold Fx®) has been licensed in 2007 as a natural health product to 'help reduce the frequency, severity and duration of cold and flu symptoms by boosting the immune system' with an estimated annual sale of over \$48M [4, 5]. Our findings on the paradoxical effects of AQ and PS extract on macrophage function *in vivo* may have significant implication in the use of American ginseng polysaccharides in several clinical applications.

The structure and biological activities of the polysaccharides from the roots, leaves and fruits of *Panax ginseng* have recently been reviewed by Sun [2]. There is limited information on American ginseng. Our study *in vitro* has demonstrated the up-regulation of inflammatory mediators production by AQ and crude PS extracts in rat alveolar macrophages (Figure 13) which validated what we have previously reported [3 and 6]. In addition we have demonstrated specificity of PS in that acidic but not neutral species of the PS was bioactive. Following sub-acute oral administration, both PS and AQ extracts were also immuno-stimulatory based on elevation of plasma cytokine levels and increase in the function of alveolar macrophages *ex vivo*. The immuno-stimulatory dosage used in the present *in vivo* study was comparable to those proven to be effective for cardiovascular health and for protection against diabetic retinopathy, neuropathy and cardiomyopathy reported by other investigators using identical ginseng extracts and in the same animal species [17-19]. However, the magnitude of the immunostimulatory effect *in vivo* was smaller than the *in vitro* response. This may be related to the lower bioavailability of the orally-administered ginseng extract. The pharmacokinetics of oral ginseng PS is not known, but the recovery of major ginsenosides in plasma after oral administration was quite low [20]. Result of our *in vitro* and *in vivo* studies was supportive of what was reported for CVT-E002 (a patented, poly-furanosyl-pyranosyl polysaccharide-rich extract of the root of North American ginseng): stimulation of normal mouse spleen cells and immunoglobulin G production as well as activation of peritoneal exudate macrophages leading to enhanced cytokine stimulation in treated mice.

In addition to the well-recognized immuno-stimulatory activity of ginseng, an antiinflammatory effect was shown in the present study as reflected in the reduced responsiveness of alveolar macrophages collected from ginseng-treated animals to LPS challenges *ex vivo* (Figure 10). This apparent anti-inflammatory effect of ginseng PS is different from what we have previously reported for a specific component(s) of the alcoholic extract of American ginseng as well as the anti-inflammatory effects that have been ascribed to some ginsenosides and their metabolites [6, 21-22]. This potential anti-inflammatory mechanism is being validated by evaluating changes in LPS–induced inflammatory response following polysaccharide pretreatment *in vivo* in our on-going research. The intent is to determine whether ginseng PS causes desensitization of immune cells as reported for LPS [23]. This action of ginseng, when proven, may be particularly relevant to bacterial infection and related toxicemia. An anti-inflammatory effect of ginseng polysaccharide has been reported by Zhao et al. using a model of auto-immune disease, as evidenced by the reduction in the expression of TNF-α and IFN-γ in lymphocytes in the enteric mucosal immune system of rats with collagen induced arthritis [24]. In view of the diverse immunomodulatory effects of ginseng polysaccharide, the identification of specific polysaccharides with unique property and biological action will be of great interest.

It appears that AQ and PS ginseng extracts have a paradoxical effect on macrophage function: stimulation under normal condition, but reduction when the biological system is under a pro-inflammatory state. In the context of sepsis, AQ and PS immuno-stimulatory effect will be beneficial as a first line of defense during the initial infection stage of bacterial infection by rendering macrophages to be cytotoxic [25] whereas the immune-suppressive activity may be effective in antagonizing the cytokine storm at the later stages of infection by suppressing LPS activation of macrophages.

Carbohydrates analysis represents a major challenge in analytical chemistry since neutral or acidic saccharides (mono, oligo and poly) have little UV activity. The refractive index (RI) detector, which is commonly used in HPLC analysis, has issues with baseline stability and sensitivity. The ELSD used in the present study has the advantage of its independence of any optical properties in the solutes of interest. Our HPLC-based analysis allowed the measurement of 7 mono-saccharides within a 30 minute total run time. The monosaccharide composition of *P. quinquefolius* reported in this study was similar to that described previously [3] with the addition of galacturonic acid. The monosaccharide composition provides insight into the types of polysaccharides which may be found in *P. quinquefolius*. Polysaccharides structures in *P. quinquefolius* have not previously been thoroughly studied, though several polysaccharides have been isolated from *P. ginseng* and *P. notoginseng*. Glucose and galacturonic acid were the most prominent monosaccharides detected. Previously, polysaccharide fractions from *P. ginseng* with high levels of glucose have been determined to contain starch-like glucans and arabinogalactans and fractions with high levels of galacturonic acid have been shown to contain pectins with several linked galacturonic acid domains [26]. It is possible that polysaccharides similar to these may be present in Ontario-grown American ginseng though further work will have to be done to characterize their structures.

Polysaccharides are very complex with a wide range of MW, varying monosaccharide composition and conformation (degree of branching or linearity), which contribute to their diverse structure and biological activities, these can also hamper the study of their structure–function relationships [16]. The multi-detector GPC instrument provided additional information on the polysaccharide structure. This instrument uses Triple detection with a concentration detector (refractive index detector), viscometer and light scattering detector, with each detector providing different although complementary information [20]. Pullulan standards of up to 1,000 kDa showed that the measured ginseng polysaccharide molecular weights for the crude, water soluble and deproteinated extracts are within the range of standards. The light scattering detector, which is based on fluctuations in interference between macromolecules (e.g. polysaccharides) scattering a coherent monochromatic laser beam, is considered an absolute detector for Mw values, confirming the reported values of Table 1. The viscometer is sensitive to branching effects, although the trace in Figure 9 shows no significant branching effect for the ginseng polysaccharides. The RI detector was rather sensitive to the carbohydrate fractions, showing 3 major polysaccharide components for each of the three measured extracts. The high molecular weight peak has a very low intensity for the PTF sample, indicating that the deproteination step removed the high molecular weight fraction. The neutral extract gave similar molecular weights as the acidic polysaccharide extract, although with a higher sample recovery.
