Identification of metabolites in plasma related to different biological activities of Panax ginseng and American ginseng.

RATIONALE: Panax ginseng (PG) and American ginseng (AMG) are both medicinal plants of the Panax genus in the Acanthopanax family. Although PG and AMG have similar components of ginsenosides, there are many differences of their bioactivities. In this study, the biochemical mechanisms of different bioactivities of PG and AMG were explored by researching the differential metabolites in plasma after administration of each of PG and AMG. METHODS: In order to explore the material basis of differential bioactivities, two groups of mice were administrated orally with PG and AMG, and the method of metabolomics was used to identify the differential metabolites in plasma. Then network pharmacology was used based on the differential metabolites. Afterward, the metabolite-target-pathway network of PG and AMG was constructed; thus the pathways related to different bioactivities were analyzed. RESULTS: Through principal component analysis and orthogonal projections to latent structures discriminant analysis, there were 10 differential metabolites identified in the PG group and 8 differential metabolites identified in the AMG group. Based on network pharmacology, the differential metabolites were classified and related to differential bioactivities of PG and AMG. In the PG group, there were 6 metabolites related to aphrodisiac effect and exciting the nervous system, and 5 metabolites associated with raised blood pressure. In the AMG group, 5 metabolites were classified as having the effect of inhibiting the nervous system, and 6 metabolites were related to antihypertensive effect. CONCLUSIONS: This study explored the material basis of the differential biological activities between PG and AMG, which is significant for the research of PG and AMG use and to promote human health.

Non-targeted metabonomics to investigate the differences in the properties of ginseng and American ginseng based on rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

The basic properties of herbal medicines are cold, hot, warm, and cool. The differentiation of these properties is important for the diagnosis and treatment of diseases. Ginseng and American ginseng possess opposite properties of warm and cool, respectively. At present, the mechanisms and the influence of steaming leading to the differences in their properties are not clear and require further investigation. Therefore, nontargeted metabonomics based on liquid chromatography-mass spectrometry was applied to investigate the effects of ginseng, American ginseng, and their variants on the changes in endogenous metabolites in rat urine. A total of 19 potential biomarkers were screened out and identified, of which 17, 7, and 5, were respectively related to warm, cool, and both warm and cool properties with opposite effects. The metabolic pathways corresponded to fatty acids, lipids, glycolysis, and energy metabolisms. The warm and tonic effects of red ginseng are stronger than those of ginseng and consistent with the theory of traditional Chinese medicine. The red American ginseng has cool property; however, the degree of coolness is less than that of American ginseng. This study provides a reference methodology to understand the effects of processing and mechanisms associated with the differences in the properties of herbal medicines.

177 Saponins, Including 11 New Compounds in Wild Ginseng Tentatively Identified via HPLC-IT-TOF-MSn, and Differences among Wild Ginseng, Ginseng under Forest, and Cultivated Ginseng.

Wild ginseng (W-GS), ginseng under forest (F-GS, planted in mountain forest and growing in natural environment), and cultivated ginseng (C-GS) were compared via HPLC-DAD and HPLC-IT-TOF-MSn. A total of 199 saponins, including 16 potential new compounds, were tentatively identified from 100 mg W-GS (177 saponins in W-GS with 11 new compounds), F-GS (56 saponins with 1 new compound), and C-GS (60 saponins with 6 new compounds). There were 21 saponins detected from all the W-GS, F-GS, and C-GS. Fifty saponins were only detected from W-GS, including 23 saponins found in ginseng for the first time. Contents of ginsenosides Re (12.36-13.91 mg/g), Rh1 (7.46-7.65 mg/g), Rd (12.94-12.98 mg/g), and the total contents (50.52-55.51 mg/g) of Rg1, Re, Rf, Rb1, Rg2, Rh1, and Rd in W-GS were remarkably higher than those in F-GS (Re 1.22-3.50 mg/g, Rh1 0.15-1.49 mg/g, Rd 0.19-1.49 mg/g, total 5.69-18.74 mg/g), and C-GS (Re 0.30-3.45 mg/g, Rh1 0.05-3.42 mg/g, Rd 0.17-1.68 mg/g, total 2.99-19.55 mg/g). Contents of Re and Rf were significantly higher in F-GS than those in C-GS (p < 0.05). Using the contents of Re, Rf, or Rb1, approximately a half number of cultivated ginseng samples could be identified from ginseng under forest. Contents of Rg1, Re, Rg2, Rh1, as well as the total contents of the seven ginsenosides were highest in ginseng older than 15 years, middle-high in ginseng between 10 to 15 years old, and lowest in ginseng younger than 10 years. Contents of Rg1, Re, Rf, Rb1, Rg2, and the total of seven ginsenosides were significantly related to the growing ages of ginseng (p < 0.10). Similarities of chromatographic fingerprints to W-GS were significantly higher (p < 0.05) for F-GS (median: 0.824) than C-GS (median: 0.745). A characteristic peak pattern in fingerprint was also discovered for distinguishing three types of ginseng. Conclusively, wild ginseng was remarkably superior to ginseng under forest and cultivated ginseng, with ginseng under forest slightly closer to wild ginseng than cultivated ginseng. The differences among wild ginseng, ginseng under forest, and cultivated ginseng in saponin compositions and contents of ginsenosides were mainly attributed to their growing ages.

Resolving complicated relationships of the Panax bipinnatifidus complex in southwestern China by RAD-seq data.

The P. binpinnatifidus complex included most of the Panax species distributed in Sino-Himalaya regions except for P. pseudoginseng, P. stipuleanatus and P. notoginseng. However, the delimitation and identification of these taxa within the species complex are very difficult due to the existence of morphological intermediates, and their evolutionary relationships remain unresolved despite several studies have been carried out based on traditional DNA markers. The taxonomic uncertainty hinders the identification, conservation and exploration of these wild populations of Panax. To study this species complex, we employed ddRAD-seq data of these taxa from 18 different localities of southwestern China, using two RAD analysis pipelines, STACKS and pyRAD. Based on the results of phylogenetic analysis, the species complex was divided into four clades with high supports, which largely agreed with morphologically described species. Two clades, corresponding to P. vietnamensis and P. zingiberensis, respectively, were sister groups, indicating that these two species had a closer genetic relationship; the third clade was consisted of samples with bamboo-like rhizomes named as P. wangianus clade, and the fourth one with moniliform rhizomes was named as P. bipinnatifidus clade. The population genetic structure analysis and D-statistics test showed the localized admixture among these species, which indicated that introgression had occurred among the related lineages continuously distributed in southeastern Yunnan and adjacent regions.

Functional Characterization of BRASSINAZOLE-RESISTANT 1 in Panax Ginseng (PgBZR1) and Brassinosteroid Response during Storage Root Formation.

Brassinosteroids (BRs) play crucial roles in the physiology and development of plants. In the model plant Arabidopsis, BR signaling is initiated at the level of membrane receptors, BRASSINOSTEROIDS INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) complex, thus activating the transcription factors (TFs) BRASSINAZOLE RESISTANT 1/BRI1-EMS-SUPPRESSOR 1 (BZR1/BES1) to coordinate BR responsive genes. BRASSINOSTEROIDS INSENSITIVE 2 (BIN2), glycogen synthase kinase 3 (GSK3) like-kinase, negatively regulates BZR1/BES1 transcriptional activity through phosphorylation-dependent cytosolic retention and shuttling. However, it is still unknown whether this mechanism is conserved in Panax ginseng C. A. Mayer, a member of the Araliaceae family, which is a shade-tolerant perennial root crop. Despite its pharmacological and agricultural importance, the role of BR signaling in the development of P. ginseng and characterization of BR signaling components are still elusive. In this study, by utilizing the Arabidopsisbri1 mutant, we found that ectopic expression of the gain of function form of PgBZR1 (Pgbzr1-1D) restores BR deficiency. In detail, ectopic expression of Pgbzr1-1D rescues dwarfism, defects of floral organ development, and hypocotyl elongation of bri1-5, implying the functional conservation of PgBZR1 in P. ginseng. Interestingly, brassinolide (BL) and BRs biosynthesis inhibitor treatment in two-year-old P. ginseng storage root interferes with and promotes, respectively, secondary growth in terms of xylem formation. Altogether, our results provide new insight into the functional conservation and potential diversification of BR signaling and response in P. ginseng.

Characteristics of Panax ginseng Cultivars in Korea and China.

Ginseng (Panax ginseng Meyer) is one of the most important medicinal herbs in Asia. Its pharmacological activity comes from ginsenosides, and its roots are produced commercially for traditional and Oriental medicine. Though 17 Panax species are available around the world, there was a need to develop cultivars adapted to different climatic conditions and resistant to various diseases while still producing high-quality, high-yield roots. Thus, 12 and 9 commercial P. ginseng cultivars have been registered in South Korea and China, respectively. Those varieties show superiority to local landraces. For example, Chunpoong is more highly resistant to rusty rot disease than the local Jakyungjong landrace and has a good root shape; it is highly cultivated to produce red ginseng. The Chinese cultivar Jilin Huangguo Renshen has higher ginsenoside content than its local landraces. This review provides information about P. ginseng cultivars and offers directions for future research, such as intra- and interspecific hybridization.

Ginseng Extract Ameliorates the Negative Physiological Effects of Heat Stress by Supporting Heat Shock Response and Improving Intestinal Barrier Integrity: Evidence from Studies with Heat-Stressed Caco-2 Cells, C. elegans and Growing Broilers.

Climatic changes and heat stress have become a great challenge in the livestock industry, negatively affecting, in particular, poultry feed intake and intestinal barrier malfunction. Recently, phytogenic feed additives were applied to reduce heat stress effects on animal farming. Here, we investigated the effects of ginseng extract using various in vitro and in vivo experiments. Quantitative real-time PCR, transepithelial electrical resistance measurements and survival assays under heat stress conditions were carried out in various model systems, including Caco-2 cells, Caenorhabditis elegans and jejunum samples of broilers. Under heat stress conditions, ginseng treatment lowered the expression of HSPA1A (Caco-2) and the heat shock protein genes hsp-1 and hsp-16.2 (both in C. elegans), while all three of the tested genes encoding tight junction proteins, CLDN3, OCLN and CLDN1 (Caco-2), were upregulated. In addition, we observed prolonged survival under heat stress in Caenorhabditis elegans, and a better performance of growing ginseng-fed broilers by the increased gene expression of selected heat shock and tight junction proteins. The presence of ginseng extract resulted in a reduced decrease in transepithelial resistance under heat shock conditions. Finally, LC-MS analysis was performed to quantitate the most prominent ginsenosides in the extract used for this study, being Re, Rg1, Rc, Rb2 and Rd. In conclusion, ginseng extract was found to be a suitable feed additive in animal nutrition to reduce the negative physiological effects caused by heat stress.

Novel Foveavirus (the family Betaflexiviridae) species identified in ginseng (Panax ginseng).

Ginseng (Panax ginseng) is a valuable herb that is widely cultivated in Korea, China, and Japan because it contains a variety of pharmacologically active substances with a wide range of positive effects on human health. Identification and prevention of disease-causing viral pathogens of ginseng is important for improving the yield and quality of ginseng-derived bioactive molecules. In this study, the genome sequence of the virus Panax ginseng flexivirus 1 (PgFV1) was identified from a ginseng root transcriptome data set. Sequence comparison and phylogenetic analysis showed that PgFV1 is a novel plant RNA virus species of the genus Foveavirus (the family Betaflexiviridae). Foveaviruses have flexuous and filamentous virions with a single-stranded positive-sense mono-segmented RNA genome. Its infection causes diseases with mosaic and ringspot symptoms in the stems and leaves. The PgFV1 genome encodes for 5 open reading frames: a replicase polyprotein for viral genome replication, 3 triple gene block proteins for viral cell-to-cell movement, and coat protein. Phylogenetic trees inferred from replicase polyprotein or coat protein sequences showed that PgFV1 is most closely related to grapevine virus T. PgFV1 is the first foveavirus identified to be associated with ginseng. Given the potential pathogenic features of previously known foveaviruses and importance of ginseng in the health industry, the PgFV1 genome sequence may be highly useful for studying ginseng foveaviruses. Keywords: ginseng; Panax ginseng flexivirus 1; Foveavirus; Betaflexiviridae.

Black Ginseng and Its Saponins: Preparation, Phytochemistry and Pharmacological Effects.

Black ginseng is a type of processed ginseng that is prepared from white or red ginseng by steaming and drying several times. This process causes extensive changes in types and amounts of secondary metabolites. The chief secondary metabolites in ginseng are ginsenosides (dammarane-type triterpene saponins), which transform into less polar ginsenosides in black ginseng by steaming. In addition, apparent changes happen to other secondary metabolites such as the increase in the contents of phenolic compounds, reducing sugars and acidic polysaccharides in addition to the decrease in concentrations of free amino acids and total polysaccharides. Furthermore, the presence of some Maillard reaction products like maltol was also engaged. These obvious chemical changes were associated with a noticeable superiority for black ginseng over white and red ginseng in most of the comparative biological studies. This review article is an attempt to illustrate different methods of preparation of black ginseng, major chemical changes of saponins and other constituents after steaming as well as the reported biological activities of black ginseng, its major saponins and other metabolites.

Analysis of Ginsenoside Content (Panax ginseng) from Different Regions.

Recently Panax ginseng has been grown as a secondary crop under a pine tree canopy in New Zealand (NZ). The aim of the study is to compare the average content of ginsenosides from NZ-grown ginseng and its original native locations (China and Korea) grown ginseng. Ten batches of NZ-grown ginseng were extracted using 70% methanol and analyzed using LC-MS/MS. The average content of ginsenosides from China and Korea grown ginseng were obtained by collecting data from 30 and 17 publications featuring China and Korea grown ginseng, respectively. The average content of total ginsenosides in NZ-grown ginseng was 40.06 ± 3.21 mg/g (n = 14), which showed significantly (p < 0.05) higher concentration than that of China grown ginseng (16.48 ± 1.24 mg/g, n = 113) and Korea grown ginseng (21.05 ± 1.57 mg/g, n = 106). For the individual ginsenosides, except for the ginsenosides Rb2, Rc, and Rd, ginsenosides Rb1, Re, Rf, and Rg1 from NZ-grown ginseng were 2.22, 2.91, 1.65, and 1.27 times higher than that of ginseng grown in China, respectively. Ginsenosides Re and Rg1 in NZ-grown ginseng were also 2.14 and 1.63 times higher than ginseng grown in Korea. From the accumulation of ginsenosides, New Zealand volcanic pumice soil may be more suitable for ginseng growth than its place of origin.

Identification of certain Panax species to be potential substitutes for Panax notoginseng in hemostatic treatments.

Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow (P. notoginseng) is a highly valued Chinese materia medica having a hemostatic effect and mainly used for the treatment of trauma and ischemic cardiovascular diseases. Stringent growth requirements, weak resistance to insect pests and plant diseases, arsenic contamination and continuous cropping constitute hurdles to further increases in the agricultural production of P. notoginseng. This review focuses on the traditional uses (based on traditional Chinese medicine theory), major chemical components, biological activities, pharmacological properties, geographical distributions and historical development of taxonomy of P. notoginseng and its related species in Panax genus, including Panax japonicus C. A. Meyer (P. japonicus), Panax japonicus C. A. Meyer var. major (Burkill) C. Y. Wu et K. M. Feng (P. japonicus var. major) and Panax japonicus C. A. Meyer var. bipinnatifidus (Seem.) C. Y. Wu et K. M. Feng (P. japonicus var. bipinnatifidus) are reviewed. This review sheds light on the origin herbs of Zhujieshen (ZJS) and Zhuzishen (ZZS), e.g., P. japonicas var japonicas, P. japonicus var. major and P. japonicus var. bipinnatifidus could be used as a substitute for P. notoginseng as hemostatic herbs.

An Integrated LC-MS-Based Strategy for the Quality Assessment and Discrimination of Three Panax Species.

: The quality assessment and discrimination of Panax herbs are very challenging to perform due to the complexity and variability of their chemical compositions. An integrated strategy was established using UHPLC-Q-Exactive/HRMS and HPLC-ESI-MS/MS to achieve an accurate, rapid, and comprehensive qualitative and quantitative analysis of Panax japonicas (PJ), Panax japonicus var. major (PM), and Panax zingiberensis (PZ). Additionally, discrimination among the three species was explored with partial least squares⁻discriminant analysis (PLS-DA) and orthogonal partial least squares⁻discriminant analysis (OPLS-DA) score plots. A total of 101 compounds were plausibly or unambiguously identified, including 82 from PJ, 78 from PM, and 67 from PZ. Among them, 16 representative ginsenosides were further quantified in three herbs. A clear discrimination between the three species was observed through a multivariate statistical analysis on the quantitative data. Nine compounds that allowed for discrimination between PJ, PM, and PZ were discovered. Notably, ginsenoside Rf (G-Rf), ginsenoside F3 (G-F3), and chikusetsu saponin IV (CS-IV) were the three most important differential compounds. The research indicated that the integrated LC-MS-based strategy can be applied for the quality assessment and discrimination of the three Panax herbs.

Comparison of Serum Metabolite Changes of Radiated Mice Administered with Panax quinquefolium from Different Cultivation Regions Using UPLC-Q/TOF-MS Based Metabolomic Approach.

Chemometric analysis of bioactive compounds revealed that American ginsengs (AGs) from different cultivation regions of China had a difference in quality, which indicates their possible pharmacological difference. A UPLC-Q/TOF-MS-based untargeted metabolomic approach was used to uncover serum metabolite changes in radiated mice pre-administered with AG root decoctions from seven cultivation regions and to further assess their quality difference. OPLS-DA revealed that 51 metabolites (ESI−) and 110 (ESI⁺) were differentially expressed in sera between the control and the radiated model mice. Heatmap analysis further revealed that AG could not reverse most of these radiation-altered metabolites, which indicates dietary supplement of AG before cobalt radiation had the weak potential to mediate serum metabolites that were altered by the sub-lethal high dose radiation. In addition, 83 (ESI−) and 244 (ESI⁺) AG altered metabolites were detected in radiated mice under radiation exposure. Both OPLS-DA on serum metabolomes and heatmap analysis on discriminant metabolites showed that AGs from different cultivation regions differentially influenced metabolic alterations in radiated mice, which indicates AGs from different cultivation regions showed the pharmacological difference in modulation of metabolite changes. AGs from Shandong, Shanxi, and Beijing provinces had more similar pharmacological effects than AGs from USA, Canada, Jilin, and Heilongjiang. Finally, 28 important potential biomarkers were annotated and assigned onto three metabolic pathways including lipid, amino acid, and energy metabolisms.

[Development of indel markers for molecular authentication of Panax ginseng and P. quinquefolius].

Panax ginseng and P. quinquefolius are two kinds of important medicinal herbs. They are morphologically similar but have different pharmacological effects. Therefore, botanical origin authentication of these two ginsengs is of great importance for ensuring pharmaceutical efficacy and food safety. Based on the fact that intron position in orthologous genes is highly conserved across plant species, intron length polymorphisms were exploited from unigenes of ginseng. Specific primers were respectively designed for these two species based on their insertion/deletion sequences of cytochrome P450 and glyceraldehyde 3-phosphate dehydrogenase, and multiplex PCR was conducted for molecular authentication of P.ginseng and P. quinquefolius. The results showed that the developed multiplex PCR assay was effective for molecular authentication of P.ginseng and P. quinquefolius without strict PCR condition and the optimization of reaction system.This study provides a preferred ideal marker system for molecular authentication of ginseng,and the presented method can be employed in origin authentication of other herbal preparations.

Intercontinental and intracontinental biogeography of the eastern Asian - Eastern North American disjunct Panax (the ginseng genus, Araliaceae), emphasizing its diversification processes in eastern Asia.

The intercontinental biogeography between eastern Asia and eastern North America has attracted much attention from evolutionary biologists. Further insights into understanding the evolution of the intercontinental disjunctions have been hampered by the lack of studies on the intracontinental biogeography in eastern Asia, a region with complex geology, geography, climates and habitats. Herein we studied the biogeographic history of the eastern Asian-eastern North American disjunct genus Panax with special emphasis on the investigation of its uneven diversification in Asia. This study reconstructs the diversification history of Panax and also emphasizes a large clade of Panax taxa, which has a wide distribution in eastern Asia, but was unresolved in previous studies. We examined the noncoding plastid DNA fragments of trnH-psbA, rps16, and psbM-trnD, the mitochondrial b/c intron of NAD1, and the nuclear ribosomal internal transcribed spacer (ITS) region of 356 samples from 47 populations. The results revealed the subtropical Northern Hemisphere origin (Asia or Asia and North America) of Panax in the Paleocene. Intercontinental disjunctions between eastern Asia and eastern North America formed twice in Panax, once estimated in early Eocene for the split of P. trifolius and another in mid-Miocene for the divergence of P. quinquefolius. Intercontinental diversifications in Panax showed temporal correlation with the increase of global temperature. The evolutionary radiation of the P. bipinnatifidus species complex occurred around the boundary of Oligocene and Miocene. Strong genetic structure among populations of the species complex was detected and the populations may be isolated by distance. The backbone network and the Bayesian clustering analysis revealed a major evolutionary radiation centered in the Hengduan Mountains of western China. Our results suggested that the evolutionary radiation of Panax was promoted by geographic barriers, including mountain ranges (Hengduan Mountains, Nanling Mountains and Wuyishan Mountains), oceans and altitudinal shifts, which further contribute to the knowledge of the uneven species diversification between eastern Asia and North America.

[Identification of Panax ginseng, P. notoginseng and P. quinquefolius admixture by multiplex allele-specific polymerase chain reaction].

To achieve a molecular method to identify Panax ginseng, P. notoginseng,P. quinquefolius and their admixture. The ITS,18S and matK sequences of Panax genus were analyzed to develop species-specific SNP marker. Three pairs of species-specific primers were designed to establish a multiplex allele-specific polymerase chain reaction (MAS-PCR) and the samples from different region were tested. The results showed that when the annealing temperature was 60 ℃ and the cycle number was 35, approximately 250, 500,1 000 bp specific band were obtained from P. ginseng, P. notoginseng and P. quinquefolius obtain, respectively. This method could also be used to authentificate admixture samples and could detect 0.5% percent of P. notoginseng or P. quinquefolius adulterated in P. ginseng, or 0.5% percent of P. ginseng or P. quinquefolius adulterated in P. notoginseng. The detect limit of P. ginseng in P. quinquefolius was 0.5% and P. notoginseng in P. quinquefolius was 1%. This results showed that the present method could be used as a promise method to identify Panax ginseng, P. notoginseng, P. quinquefolius and their admixture.

Development of interspecies hybrids to increase ginseng biomass and ginsenoside yield.

KEY MESSAGE: Interspecific hybrids between Panax ginseng and P. quinquefolius results in hybrid vigor and higher ginsenoside contents. Ginseng is one of the most important herbs with valued pharmaceutical effects contributing mainly by the presence of bioactive ginsenosides in the roots. However, ginseng industry is impeded largely by its biological properties, because ginseng plants are slow-growing perennial herbs with lower yield. To increase the ginseng yield and amounts of ginsenosides, we developed an effective ginseng production system using the F(1) progenies obtained from the interspecific reciprocal cross between two Panax species: P. ginseng and P. quinquefolius. Although hybrid plants show reduced male fertility, F(1) hybrids with the maternal origin either from P. ginseng or P. quinquefolius displayed heterosis; they had larger roots and higher contents of ginsenosides as compared with non-hybrid parental lines. Remarkably, the F(1) hybrids with the maternal origin of P. quinquefolius had much higher ginsenoside contents, especially ginsenoside Re and Rb1, than those with the maternal origin of P. ginseng. Additionally, non-targeted metabolomic profiling revealed a clear increase of a large number of primary and secondary metabolites including fatty acids, amino acids and ginsenosides in hybrid plants. To effectively identify the F(1) hybrids for the large-scale cultivation, we successfully developed a molecular marker detection system for discriminating F(1) reciprocal hybrids. In summary, this work provided a practical system for reciprocal hybrid ginseng production, which would facilitate the ginseng production in the future.

High-performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia.

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

A simple real-time polymerase chain reaction (PCR)-based assay for authentication of the Chinese Panax ginseng cultivar Damaya from a local ginseng population.

Panax ginseng is one of the most important medicinal plants in the Orient. Owing to its increasing demand in the world market, cultivated ginseng has become the main source of medicinal material. Among the Chinese ginseng cultivars, Damaya commands higher prices and is grown in significant proportions among the local ginseng population. Due to the lack of rapid and accurate authentication methods, Damaya is distributed among different cultivars in the local ginseng population in China. Here, we identified a unique, Damaya-specific single nucleotide polymorphism (SNP) site present in the second intron of mitochondrial cytochrome c oxidase subunit 2 (cox2). Based on this SNP, a Damaya cultivar-specific primer was designed and an allele-specific polymerase chain reaction (PCR) was optimized for the effective molecular authentication of Damaya. We designed a method by combining a simple DNA isolation method with real-time allele-specific PCR using SYBR Green I fluorescent dye, and proved its efficacy in clearly discriminated Damaya cultivar from other Chinese ginseng cultivars according to the allelic discrimination analysis. Hence, this study provides a simple and rapid assay for the differentiation and conservation of Damaya from the local Chinese ginseng population.

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication.

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to "Gopoong" and "K-1" were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information.