Anti-hyperglycemic effects of ginseng: Comparison between root and berry

Previous studies demonstrated that both ginseng root and ginseng berry possess anti-diabetic activity. However, a direct comparison between the root and the berry under the same experimental conditions has not been conducted. In the present study, we compared anti-hyperglycemic effect between Panax ginseng root and Panax ginseng berry in ob/ob mice, which exhibit profound obesity and hyperglycemia that phenotypically resemble human type-2 diabetes. We observed that ob/ob mice had high baseline glucose levels (195 mg/dl). Ginseng root extract (150 mg/kg body wt.) and ginseng berry extract (150 mg/kg body wt.) significantly decreased fasting blood glucose to 143 +/- 9.3 mg/dl and 150 +/- 9.5 mg/dl on day 5, respectively (both P < 0.01 compared with the vehicle). On day 12, although fasting blood glucose level did not continue to decrease in the root group (155 +/- 12.7 mg/dl), the berry group became normoglycemic (129 +/- 7.3 mg/dl; P < 0.01). We further evaluated glucose tolerance using the intraperitoneal glucose tolerance test. On day 0, basal hyperglycemia was exacerbated by intraperitoneal glucose load, and failed to return to baseline after 120 min. After 12 days of treatment with ginseng root extract (150 mg/kg body wt.), the area under the curve (AUC) showed some decrease (9.6%). However, after 12 days of treatment with ginseng berry extract (150 mg/kg body wt.), overall glucose exposure improved significantly, and the AUC decreased 31.0% (P < 0.01). In addition, we observed that body weight did not change significantly after ginseng root extract (150 mg/kg body wt.) treatment, but the same concentration of ginseng berry extract significantly decreased body weight (P < 0.01). These data suggest that, compared to ginseng root, ginseng berry exhibits more potent anti-hyperglycemic activity, and only ginseng berry shows marked anti-obesity effects in ob/ob mice.     

Saponin production by hairy root cultures ofPanax ginseng CA meyer: Influence of PGR and polyamines

A culture of hairy roots ofPanax ginseng C.A. Meyer was set up in order to investigate the possibility of producing ginseng saponin. Roots cultured in 1/2 MS medium in the presence of 2 mg/L IAA and 0.1 mM spermidine showed the maximal growth rate, whereas other polyamines increased the growth of hairy roots only slightly or not at all. High saponin root contents were obtained in culture media supplemented with 0.5 mg/L GA and 1 mM putrescine.     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

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.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

In vitro metabolism of ginsenosides by the ginseng root pathogen Pythium irregulare

The role of ginseng saponins (ginsenosides) as modulators or inhibitors of disease is vague, but our earlier work supports the existence of an allelopathic relationship between ginsenosides and soilborne microbes. Interestingly, this allelopathy appears to significantly promote the growth of the important ginseng pathogen, Pythium irregulare while inhibiting that of an antagonistic non-pathogenic fungus, Trichoderma hamatum. Herein we report on the apparent selective metabolism of 20(S)-protopanaxadiol ginsenosides by an extracellular glycosidase from P. irregulare. Thus, when P. irregulare was cultured in the presence of a purified (> 90%) ginsenoside mixture, nearly all of the 20(S)-protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and to a limited extent G-XVII) were metabolized into the minor ginsenoside F2, at least half of which appears to be internalized by the organism. No metabolism of the 20(S)-protopanaxatriol ginsenosides (Rg1 and Re) was evident. By contrast, none of the ginsenosides added to the culture medium of the non-pathogenic fungus T. hamatum were metabolized. The metabolism of 20(S)-protopanaxadiol ginsenosides by P. irregulare appears to occur through the hydrolysis of terminal monosaccharide units from disaccharides present at C-3 and/or C-20 of ginsenosides Rb1, Rc, Rb2, Rd and G-XVII to yield one major product, ginsenoside F2 and one minor product (possibly G-III). A similar transformation of ginsenosides was observed using a crude protein preparation isolated from the spent medium of P. irregulare cultures.     

RAPD analysis of genome variability of planted ginseng,Panax ginseng

Genome variability of 23 ginseng plants (Panax ginseng) grown in culture in Primorskii Krai was studied by RAPD method. Eleven arbitrary chosen primers were used to analyze 138 loci of DNA samples, 17 of which appeared to be polymorphic. The OPD-11-1000 fragment was found to be a RAPD marker allowing plants to be differentiated according to their morphotype. Using five primers, it was demonstrated that the genetic polymorphism of the cultivated plants is lower than that in nature (7.6% and 10.6%, respectively). Dendrograms of genetic relatedness are in accord with genetic differences between individuals of plantedP. ginseng belonging to different morphotypes, and demonstrate close relatedness of one of the morphotypes to wild plants. This morphotype could be recommended for reintroduction into natural habitats.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

Characterization of soybean vegetative storage proteins and genes

Summary Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5-flanking regions. A second genomic clone that was studied contained the 5 regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.Cooperative research between USDA/Agricultural Research Service and the Indiana Agricultural Experiment Station. Journal Paper No. 12,192 from the Indiana Agricultural Experiment Station     

Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis

To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Characterization of a T-DNA insertion mutant for the protein import receptor atToc33 from chloroplasts

In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.Communicated by R. Hagemann     

Construction of a BAC library of Korean ginseng and initial analysis of BAC-end sequences

We estimated the genome size of Korean ginseng ( Panax ginseng C.A. Meyer), a medicinal herb, constructed a Hin dIII BAC library, and analyzed BAC-end sequences to provide an initial characterization of the library. The 1C nuclear DNA content of Korean ginseng was estimated to be 3.33 pg (3.12×10³ Mb). The BAC library consists of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents. Sequencing of 2167 BAC clones generated 2492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2%, 20.9% and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. A comparison of the functional categories represented by the protein-coding regions found in BAC-end sequences with those of Arabidopsis revealed that proteins pertaining to energy metabolism, subcellular localization, cofactor requirement and transport facilitation were more highly represented in the P. ginseng sample. In addition, a sequence encoding a glucosyltransferase-like protein implicated in the ginsenoside biosynthesis pathway was also found. The majority of the transposable element sequences found belonged to the gypsy type (67.6%), followed by copia (11.7%) and LINE (8.0%) retrotransposons, whereas DNA transposons accounted for only 2.1% of the total in our sequence sample. Higher levels of transposable elements than protein-coding regions suggest that mobile elements have played an important role in the evolution of the genome of Korean ginseng, and contributed significantly to its complexity. We also identified 103 microsatellites with 3–38 repeats in their motifs. The BAC library and BAC-end sequences will serve as a useful resource for physical mapping, positional cloning and genome sequencing of P. ginseng.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

Pulse treatments of penicillin-G and streptomycin minimise internal infections and have post-treatment effects on the morphogenesis of ginseng root culture

This study was initiated to determine whether antibiotic pulse treatments (APT) could effectively eliminate internal infections of ginseng (Panax ginseng) root explants containing vascular tissue, and subsequently have post-treatment effects on changing explant behaviors in callus induction and organogenesis or somatic embryogenesis. For contamination control, a treatment of 40 min with an antibiotic solution consisting of 1000 mg/1 of penicillin-G and 1000 mg/1 of streptomycin immediately following Na-hypochlorite sterilisation significantly decreased contamination rate. Extending treatment time to 2–3 h further lowered the contamination rate to 30–40%. On the other hand, explants treated with antibiotics for 20 min or less were all contaminated. APT also had post-treatment effects; it delayed callus induction for 1–12 months depending on pulse duration and stimulated the generation of more hardand darker looking than fragile- and lighter looking callus. The induced callus proliferated at a depressed rate, increasing subculture intervals from 1 to several weeks, and not until after five subcultures did it fully recover. The regeneration ability of the recovered callus was also affected by APT: the regeneration of adventitious roots was promoted, whereas somatic embryos were not observed.Abbreviations APT Antibiotic pulse treatments - 2,4-D 2,4-dichlorophenoxyacetic acid     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

A pilot-scale culture of multiple adventitious roots of ginseng was established using a balloon-type bubble bioreactor. Adventitious roots (2 cm) induced from callus were cultured in plastic Petri dishes having 20 ml of solid Schenk and Hildebrandt (1972) medium containing 3% sucrose, 0.15% gelrite, and 24.6 μM indole-3-butric acid. An average of 29 secondary multiple adventitious roots were produced after 4 weeks of culture. These secondary roots were elongated on the same medium, reaching a length of 5 cm after 6 weeks of culture. A time course study revealed that maximum yields in 5-l and 20-l bioreactors were approximately 500 g and 2.2 kg at day 42 with 60 g and 240 g inoculations, respectively. Cutting twice during the culture increased the total amount of biomass produced. The root biomass in a 20-l balloon-type bubble bioreactor was 2.8 kg at harvest with 240 g of inoculum after 8 weeks of culture. The total saponin content obtained from small-scale and pilot-scale balloon type bubble bioreactors was around 1% based on dry weight. Inoculation of 500 g fresh weight of multiple adventitious roots into a 500 l balloon-type bubble bioreactor with cutting at 4 and 6 weeks after inoculation produced approximately 74.8 kg of multiple roots. The ginsengnoside profiles of these multiple adventitious roots were similar to profiles of field-grown ginseng roots when analyzed by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.