Proteomic analysis of Korean ginseng (Panax ginseng C.A. Meyer)

Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways operant in ginsenoside biosynthesis, or the genes involved therein. Proteomics analysis is an approach to elucidate the physiological characteristics and biosynthetic pathways of ginsenosides, main components of ginseng. In this review, we introduced the recent progress in proteomics studies of ginseng (Panax ginseng C.A. Meyer). We briefly reference the genomic analyses of P. ginseng, without which proteomics approaches would have been impossible. Functional genomics studies regarding secondary metabolism in P. ginseng are also introduced here, in order to introduce possible future prospects for further study.     

RG-II from Panax ginseng C.A. Meyer suppresses asthmatic reaction

In asthma, T helper 2 (T(H)2)-type cytokines such as interleukin (IL)-4, IL-5, and IL-13 are produced by activated CD4(+) T cells. Dendritic cells played an important role in determining the fate of naive T cells into either T(H)1 or T(H)2 cells. We determined whether RG-II regulates the T(H)1/T(H)2 immune response by using an ovalbumin-induced murine model of asthma. RG-II reduced IL-4 production but increased interferon- gamma production, and inhibited GATA-3 gene expression. RG-II also inhibited asthmatic reactions including an increase in the number of eosinophils in bronchoalveolar lavage fluid, an increase in inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyperresponsiveness. This study provides evidence that RG-II plays a critical role in ameliorating the pathogenic process of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of RG-II in terms of its effects in a murine model of asthma.     

RAPD- and allozyme analysis of genetic variability of Panax ginseng C.A. Meyer and P. quinquefolius L

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

人参EST资源的SSR信息分析

从7055条人参EST序列中搜索出791个SSR,其出现频率为11．21％,平均长度为21．37bp,平均分布频率为1／5．7kb。二核苷酸重复是主要的重复类型,占全部EST-SSR的56．89％,其次是三核苷酸重复的占全部SSR的21．11％。AT、GAA是二核苷酸和三核苷酸中出现次数最多的重复基元类型,分别占28．89％和10．18％。     

Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C. A. Meyer)

Summary Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.     

Extensive characterization of peptides from Panax ginseng C. A. Meyer using mass spectrometric approach

Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS‐based data mining and sequencing. The screen on the charge states of precursor ions indicated that Ginseng is a peptide‐rich herb in comparison of a number of commonly used herbs. The Ginseng peptides were then extracted and submitted to nano‐LC‐MS/MS analysis using different fragmentation modes, including CID, high‐energy collisional dissociation, and electron transfer dissociation. Further database search and de novo sequencing allowed the identification of total 308 peptides, some of which might have important biological activities. This study illustrates the abundance and sequences of endogenous Ginseng peptides, thus providing the information of more candidates for the screening of active compounds for future biological research and drug discovery studies.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Proteome of Oriental ginseng Panax ginseng C. A. Meyer and the potential to use it as an identification tool

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild-grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two-dimensional electrophoresis (2-DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2-DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2-DE maps of different ginseng samples. The use of these "marker proteins" may help to speed up the identification process.     

Isolation and properties of arginase from a shade plant, ginseng (Panax ginseng C.A. Meyer) roots.

Arginase (EC 3.5.3.1) was purified to homogeneity from root tissues of three-year-old ginseng (Panax ginseng C.A. Meyer), shade plant, and was found to be an extraordinarily large molecule relatively stable to heat. The enzyme was decameric having a molecular mass of 352,000 Da, with an optimal temperature and pH of 60 degrees C and 9.5, respectively. Analogues of arginine could not replace it as substrate, and a cysteine residue is at or near the active site. Maximum activity was obtained with Mn(2+) and Co(2+) also activated the proteins, whereas, both agmatine and 5'-deoxy-methylthioadenosine were inhibitors. Specific activities of the enzyme in sliced ginseng roots were increased by plant hormones such as GA(3), IAA, kinetin and putrescine, whereas the activities of the purified enzyme were unaffected by putrescine. Increases in arginase activities by these plant hormones could affect metabolism of polyamine intracellularly.     

Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1

We isolated the full-length cDNA of PgCDPK2DS1 gene whose expression was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain adult plants from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.     

Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

Acclimation of hydrogen peroxide enhances salt tolerance by activating defense-related proteins in Panax ginseng C.A. Meyer

The effect of exogenously applied hydrogen peroxide on salt stress tolerance was investigated in Panax ginseng. Pretreatment of ginseng seedlings with 100 μM H₂O₂ increased the physiological salt tolerance of the ginseng plant and was used as the optimum concentration to induce salt tolerance capacity. Treatment with exogenous H₂O₂ for 2 days significantly enhanced salt stress tolerance in ginseng seedlings by increasing the activities of ascorbate peroxidase, catalase and guaiacol peroxidase and by decreasing the concentrations of malondialdehyde (MDA) and endogenous H₂O₂ as well as the production rate of superoxide radical (O₂ ^?). There was a positive physiological effect on the growth and development of salt-stressed seedlings by exogenous H₂O₂ as measured by ginseng dry weight and both chlorophyll and carotenoid contents. Exogenous H₂O₂ induced changes in MDA, O₂ ^?, antioxidant enzymes and antioxidant compounds, which are responsible for increases in salt stress tolerance. Salt treatment caused drastic declines in ginseng growth and antioxidants levels; whereas, acclimation treatment with H₂O₂ allowed the ginseng seedlings to recover from salt stress by up-regulation of defense-related proteins such as antioxidant enzymes and antioxidant compounds.     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Antifatigue Effects of Panax ginseng C.A. Meyer: A Randomised,Double-Blind,Placebo-Controlled Trial

The present study investigated the antifatigue effects of Panax ginseng C.A. Meyer in 90 subjects (21 men and 69 women) with idiopathic chronic fatigue (ICF) in a randomised, double-blind, placebo-controlled and parallel designed trial. A bespoke 20% ethanol extract of P. ginseng (1 g or 2 g day^–1) or a placebo was administered to each group for 4 weeks, and then fatigue severity was monitored using a self-rating numeric scale (NRS) and a visual analogue scale (VAS) as a primary endpoint. Serum levels of reactive oxygen species (ROS), malondialdehyde (MDA), total glutathione (GSH) contents and glutathione reductase (GSH-Rd) activity were determined. After 4-week, P. ginseng administration decreased the total NRS score, but they were not statistically significant compared with placebo (P>0.05). Mental NRS score was significantly improved by P. ginseng administrations as 20.4±5.0 to 15.1±6.5 [95% CI 2.3∼8.2] for 1 g and 20.7±6.3 to 13.8±6.2 [95% CI −0.1∼4.2] for 2 g compared with placebo 20.9±4.5 to 18.8±2.9 [95% CI 4.1∼9.9, P<0.01]. Only 2 g P. ginseng significantly reduced the VAS score from 7.3±1.3 to 4.4±1.8 [95% CI 0.7∼1.8] compared with the placebo 7.1±1.0 to 5.8±1.3 [95% CI 2.2 ∼3.7, P<0.01]. ROS and MDA levels were lowered by P. ginseng compared to placebo. P. ginseng 1 g increased GSH concentration and GSH-Rd activity. Our results provide the first evidence of the antifatigue effects of P. ginseng in patients with ICF, and we submit that these changes in antioxidant properties contribute in part to its mechanism.

Trial Registration

Clinical Research Information Service (CRIS) KCT0000048     

Three sesquiterpene hydrocarbons from the roots of Panax ginseng C.A. Meyer (Araliaceae)

The volatile constituents of the roots of Panax ginseng C.A. Meyer have been investigated after hydrodistillation and analysed by means of different analytical methods. Besides several compounds already known three sesquiterpene hydrocarbons have been isolated from the essential oil. Structure elucidation of the bicyclic panaxene as well as of the tricyclic panaginsene and ginsinsene was performed by MS and NMR. They have been identified as (1R*,2S*,5S*)-2-ethenyl-1(1-methylethenyl)-2,6,6-trimethylbicyclo[3.2.0]heptane (panaxene), (1S*,8S*,11R*)-4,7,7,11-tetramethyltricyclo[6.3.0.0(1,5)]undec-4-ene (panaginsene) und (1R*,6R*,7R*)-3,7,10,10-tetramethyltricyclo[4.3.2.0(2,6)]undec-2-ene (ginsinsene).     

In Vitro Flowering of Ginseng (Panax ginseng C. A. Meyer) Zygotic Embryos Induced by Growth Regulators

When zygotic embryos of ginseng were cultured on Murashige andSkoog's (MS) medium supplemented with all combinations of 6-benzyladenine(BA), GA₃, and ABA of 5µM each, flower buds were inducedon the medium with either BA, BA + GA₃, or BA + GA₃ + ABA. Inall cases flower buds were formed on elongated axillary branchesfrom the cotyledonary node, while the apices remained vegetative. (Received March 30, 1991; Accepted July 16, 1991)