Background: American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway. Results: In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay. Conclusions: We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.

Background: Immuno-modulatory effects of ginseng, including both immuno-stimulatory and immuno-suppressive effects, have been widely reported. This study aims to determine whether the paradoxical immuno-modulatory effect is related to unique phytochemical profiles of different North American (NA) ginseng, namely aqueous (AQ) and alcoholic (ALC) extracts. Methods: AQ and ALC extracts were prepared and their immuno-bioactivity were studied in vitro in murine macrophages (Raw 264.7) through measuring the direct stimulatory production of pro-inflammatory mediator and cytokines as well as the suppression of lipopolysaccharide (LPS)-stimulatory response by the two extracts. Gel permeation chromatography was used to fractionate and isolate phytochemicals for characterization of ginseng extracts. Results: AQ extract up-regulated the production of nitric oxide (NO), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) while ALC extract did not. ALC extract but not AQ extract suppressed LPS-induced macrophage NO and TNF-α production. These immuno-stimulatory and suppressive effects were exhibited at similar extract concentrations. Moreover, the macrophage-stimulating activity of the AQ extract was inhibited in the presence of ALC extract. Fractionation of AQ extract revealed the presence of two major peaks at 230 nm with average molecular weights of 73,000 and 37,000 Da. The first fraction had similar elution volume as the crude polysaccharide (PS) fraction isolated from the AQ extract, and it was the only bioactive species. Parallel fractionation study of ALC extract yielded similar elution profiles; however, both sub-fractions were devoid of PS. Fraction I of the ALC extract suppressed LPS-induced NO production dose-dependently. Conclusion: ALC extract of NA ginseng, which was devoid of PS, was immuno-inhibitory whereas the AQ extract, which contained PS, was immuno-stimulatory. These extract-related anti-inflammatory and pro-inflammatory effects may be considered as the Yin and Yang actions of ginseng.

Proceedings of 2014 conference presented by Natural Health Product Research Society of Canada, in conjunction with the Canadian Institute of Chinese Medicinal Research. May 13 – 16, 2014, Kelowna, BC, Canada