Ward, Jennifer R.1,*, H. David Clarke1, Jonathan Horton1, John Brock2, Jessica Burroughs1, and Nicholas Freeman1
1 Biology Department, University of North Carolina Asheville. 2 Chemistry Department,
University of North Carolina Asheville. * jrward@unca.edu
(Presented at The Future of Ginseng and Forest Botanicals Symposium, July 12-14, 2017, Morgantown, WV)
Abstract
American ginseng (Panax quinquefolius) is a threatened and economically valuable woodland herb, distributed throughout forests in eastern North America. Previous research has shown that composition of medicinal compounds, ginsenosides, and genetic profiles vary within and among western North Carolina (WNC) populations. In this study, samples were collected from 3o wild-grown American ginseng plants. Root tissue was non-destructively subsampled for ginsenoside analysis via High Performance Liquid Chromatography, and leaflet samples were collected for analysis of six DNA microsatellite regions to assess genetic diversity. A majority of WNC populations were dominated by the RG (Re/Rg1 < 1) chemotype, while three populations had individuals with I (1 < Re/Rg1 < 2) and RE (Re/Rg1 > 2) chemotypes. Composite genetic distances were not correlated with any ginsenoside measure. Future studies will use commercial seeds and wild transplants into common gardens to determine the relative contributions of genetic and environmental factors to the production of medicinally-active compounds in these plants.
Keywords: American ginseng, chemotype, genotype, ginsenoside, microsatellite
Introduction
American ginseng (Panax quinquefolius L., Araliaceae) is a perennial herb inhabiting deciduous hardwood forests from Georgia to Quebec (Anderson et al. 2002) and as far west as Oklahoma (USDA Plants 2017). The plant produces multiple compound leaves and a single umbel; the latter matures to produce berries, which can be dispersed by thrushes (Hruska et al. 2014). The species is threatened, endangered, or of special concern in multiple states throughout its range (USDA Plants 2017). Wild-harvested American ginseng roots had a market value of up to $1,400 per pound for the 2015 season, while the price for dried, cultivated roots have a steady market value of $70 to $80 a pound (Rainey 2015). The high market value for wild-harvested roots has led to overharvesting to the point of near extinction, and in 1972 the species was listed on Appendix II of the Convention on International Trade in Endangered Flora and Fauna (CITES 2017). Population viability analysis of 36 populations predicted that P. quinquefolius had a > 99% probability of going extinct in the wild within the next century (McGraw and Furedi 2005). While American ginseng has been commercially cultivated for over two hundred years, wild harvesting has continued as non-cultivated roots earn higher prices on the Asian market due to phenotypic traits (McGraw et al. 2013).
Ginseng’s secondary compounds, ginsenosides, are found within leaves and roots, and have been used in western medicine (McGraw et al. 2013). These medicinally active compounds are triterpendoid saponins, and are organized into two classes: 20(S)-protopanaxadiol, also called PPD, and t20(S)-protopanaxatriol, also called PPT. Members of the PPD class, which include Rb1, Rb2, Rc, and Rd, contain a carboxyl group on the C-6 position, while members of the PPT class, which include Re, Rg1, Rg2, and Rh1, do not (Kolodziej et al. 2013). Rb1, Rb2, Rc, Rd, Re, and Rg1 are the forms most commonly found in P. quinquefolius (Corbit et al. 2005, Schlag & McIntosh 2013). Many studies have found that Rb1 and Re are the most common ginsenosides found in American ginseng roots (Li et al. 1996, Court et al. 1996). Extracted P. quinquefolius ginsenosides have been used to treat immune, endocrine, cardiovascular, and central nervous systems disorders, and may be useful in cancer prevention (Dharmananda 2002, Corbit et al. 2005). In addition, intact American ginseng roots have been wild-harvested since the 1800s for export to the Asian market, where they are used in Traditional Chinese Medicine (TCM) (Carlson 1986).
Ginsenoside species and concentrations vary within plant organs and among individuals, with a plant’s unique suite of ginsenosides described as a chemotype. There is greater chemotypic variability in western North Carolina than in other portions of American ginseng’s range (Schlag and McIntosh 2013, Searels et al. 2013), and chemotypes can be correlated with genetic variation (Schlag and McIntosh 2013). In addition, a unique chemotype has been found in American ginseng from the southern Appalachians (Searels et al. 2013). The relative contributions of environmental, genetic, and interactive factors to this unique chemotype or to other chemotypic patterns remains poorly characterized, however. The goal of this study was to examine relationships between genetic and chemical factors in wild-collected P. quinquefolius plants from western North Carolina.
Methods
Ginsenoside Sample Collection and Preparation
A small portion of root was collected from 30 three-leaved, non-reproductive plants in western North Carolina, leaving most of the root intact. The root drying procedure mimicked commercial procedures, with wet root mass measured and samples placed in a drying oven at ~37 °C for approximately 140 hours. Dry mass was measured, and roots were ground in a Wiley Mill with a 40-mesh screen.
The extraction procedure, adapted from the methanol reflux extraction of Corbit et al. (2005), maximizes ginsenoside yield. For each sample, 100 mg of the powdered plant root was combined with 5 mL of 100% HPLC- grade methanol. Samples were refluxed at ~63 °C for 1 h, then the methanol solution was vacuum filtered through Whatman 41 Ashless filter paper. Another 5 mL of 100% HPLC-grade methanol was added to the remaining root material and allowed to reflux for 1 h. The methanol solution was filtered again through vacuum filtration and added to the previously-extracted liquid. The vacuum flask was rinsed with another 5 mL of 100% HPLC-grade methanol and added to the liquid extraction. Samples were diluted to 20 mL with 100% HPLC-grade methanol and then filtered using a 0.45 µM filter.
Ginsenoside Analysis
Standards were prepared using ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd, obtained from Indofine Chemical Company (Hillsborough, NJ). Ginsenosides in standards and plant extracts were separated by high performance liquid chromatography (HPLC, Thermo-Hypersil Gold, 150 x 3mm, C18 column 3 µm particle size, Shimadzu Inc.) using an injection volume of 20 µL with water/acetonitrile gradient elution at a rate of 0.6 mL/min. Gradient shifts were as follows:
0-22 min 95/5
22-40 min 78/22
40-50 min 55/45
50-52 min 45/55
52-58 min 35/65
The column temperature was held at 35 °C, and ultraviolet detection was set at 205 nm. Each ginsenoside was identified by retention time, which remained constant throughout the analyses. The concentration of each ginsenoside was calculated using the peak area and a six-point external standard calibration curve.
Genetic Sample Collection and Extraction
Single leaflet tissue samples were collected from 30 western North Carolina plants and stored at -80 °C until extraction. Then, whole genomic DNA was extracted from leaflets using Qiagen DNeasy Plant Mini Kits (Qiagen, Valencia, CA). DNA concentrations of samples were quantified spectrophotometrically (NanoDrop, Wilmington, DE), with ideal concentrations around 10 ng/µL. High concentrations were diluted with AE Buffer (Qiagen).
Microsatellite Amplification and Analysis
Twelve microsatellite primers specific for P. quinquefolius (Young et al. 2012) were ordered from Eurofins MWG Operon (Huntsville, AL), then screened with Polymerase Chain Reaction (PCR). The six most consistently amplifying primer sets for western North Carolina plants (B011, B119, C105, C202, D114, D227) were fluorescently-tagged then used in subsequent PCR amplifications. In each reaction, 7 µL of DNA sample was combined with 1 µL each of the forward and reverse primer (10 µM) and 9 µL of MasterMix (5 PRIME, Gaithersburg, MD). Microsatellite regions were then PCR-amplified (BIO-RAD Thermocycler, Hercules, CA) using the following protocol (Young et al. 2012):
94° C for 2 minutes
35 cycles of
94° C for 40 s
56° C for 40 s
72° C for 1 min
final extension at 72° C for 10 min
PCR products were visualized via gel electrophoresis (1% agarose gels), and successful products were multiplexed with the LIZ 500 ladder and sent to the DNA Analysis Facility at Yale University for fragment analysis. Peak calls for raw data were made in Geneious 10.2.2 with the Microsatellite 1-4-4 plugin.
Statistical Analysis
Statistical analyses were conducted in R 3.1. Composite genotypes were generated with Polysat v. 3.1.3. Euclidean distances among ginsenosides were then calculated in Vegan 2.3-5. Vegan 2.3-5 was used to conduct Mantel tests, with Spearman’s rank correlations and 9999 iterations, to discern relationships between composite genotypes and ginsenoside patterns.
Results
Analyses of data for these 30 plants revealed no relationships between genetic and ginsenoside patterns. When individual ginsenosides were used to generate composite distances, they were not related to composite genotypes (Mantel r = 0.02485, P = 0.31); neither was total ginsenoside concentration (Mantel r = 0.02324, P = 0.33). Geneotype was also not related to chemotype (Re/Rg1 ratio: Mantel r = 0.0088, P = 0.41; Rg1 concentration: Mantel r = 0.0058, P = 0.43; Re concentration: Mantel r = 0.013, P = 0.38).
Discussion
Chemotypic diversity in western North Carolina plants was depressed relative to plants in Maryland (Schlag and McIntosh 2013). This could be due to higher rates of harvesting here, or more consistent environmental conditions among sites that we sampled. This pattern might also be attributed to reduced genetic diversity in western North Carolina populations, although methodological differences between our study and that of Schlag and McIntosh (2013) render direct comparisons impossible.
Preliminary examinations of small numbers of wild-grown American ginseng from western North Carolina showed no relationships between chemical (6 individual ginsenosides) and genetic (composite genotypes with 6 microsatellite loci) properties. Perhaps ginsenoside differences are not correlated with the neutral loci discerned through microsatellite analyses. Alternatively, environmental conditions could exert more control over chemotypic patterns than innate genetic differences.
Future research will require more exhaustive sampling and analysis of these populations as well as additional populations from individuals within western North Carolina. It would also be informative to include a data from commercial seeds grown under field conditions. Finally, as contribution of environmental factors to chemotypic patterns remains uncharacterized, growing different genotypes in common gardens will allow the relative contributions of genetic and environmental factors to be discerned.
References
Anderson RC, Anderson MR, Houseman G (2002) Wild American Ginseng. Native Plants Journal 3(2):93-105.
Carlson AW (1986) Ginseng: America’s botanical drug connection to the Orient. Economic Botany 40: 233-249.
CITES (Convention on International Trade in Endangered Species) (2017) www.cites.org
Corbit RM, Ferreira JFS, Ebbs SD, Murphy LL (2005) Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) for high-performance liquid chromatography−ultraviolet analysis. Journal of Agricultural and Food Chemistry 53: 9867-9873.
Court WA, Hendel JG, Elmi J (1996) Reversed-phase high-performance liquid chromatographic determination of ginsenosides of Panax quinquefolius. Journal of Chromatography 755: 11-17.
Dharmananda S (2002) The nature of ginseng: traditional use, modern research, and the question of dosage. HerbalGram 54:34-51.
Hruska A, Souther S, McGraw J (2014) Songbird dispersal of American ginseng (Panax quinquefolius). Ecoscience 21(1):46-55.
Kołodziej B, Kowalski R, Hołderna-Kędzia E (2013). Chemical composition and chosen bioactive properties of Panax quinquefolius extracts. Panax quinquefolius Chemine Studetis Ir Kai Kurios Bioaktyvumo Savybes 24(2):151-159.
Li TSC, Mazza G, Cottrell AC, Gao L (1996) Ginsenosides in roots and leaves of American ginseng. Journal of Agricultural and Food Chemistry 44: 717–720.
McGraw JB, Furedi MA (2005) Deer browsing and population viability of a forest understory plant. Science 307(5711):920-922.
McGraw JB, Lubbers AE, Van der Voot M, Mooney EH, Furedi MA, Souther S, Turner JB, Chandler J (2013) Ecology and conservation of ginseng (Panax quinquefolius) in a changing world. Annals of the New York Academy of Sciences 1286: 62-91.
Rainey C. (2015) The Craze for Wild Ginseng, America’s Alt-Viagra. New York Magazine: New York Magazine.
Schlag EM, McIntosh MS (2013) The relationship between genetic and chemotypic diversity in American ginseng (Panax quinquefolius L.). Phytochemistry 93: 96-104.
Searels JM, Keen KD, Horton JL, Clarke HD, Ward JR (2013) Comparing ginsenoside production in leaves and roots of wild American ginseng (Panax quinquefolius). American Journal of Plant Sciences. American Journal of Plant Sciences 4:1252-1259.
USDA, NRCS. 2015. PLANTS Database (http://plants.usda.gov). National Plant Data Team Greensboro, NC 27401-4901 USA.
Young JA, Eackles MS, Springmann MJ (2012) Development of tri- and tetra- nucleotide polysomic microsatellite markers for characterization of American ginseng (Panax quinquefolius L.) genetic diversity and population structuring. Conservation Genetic Resources 4:833-836.
