Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

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.

Abstract

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 F₁ progenies obtained from the interspecific reciprocal cross between two Panax species: P. ginseng and P. quinquefolius. Although hybrid plants show reduced male fertility, F₁ 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 F₁ 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 F₁ hybrids for the large-scale cultivation, we successfully developed a molecular marker detection system for discriminating F₁ reciprocal hybrids. In summary, this work provided a practical system for reciprocal hybrid ginseng production, which would facilitate the ginseng production in the future.

     

Co-transformation of <Emphasis Type="Italic">Panax</Emphasis> major ginsenosides Rb<Subscript>1</Subscript> and Rg<Subscript>1</Subscript> to minor ginsenosides C–K and F<Subscript>1</Subscript> by <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis>

Rb₁ and Rg₁ are the major ginsenosides in protopanaxadiol and protopanaxatriol. Their content in ginsenosides was 23.8 and 17.6%, respectively. A total of 22 isolates of β-glucosidase producing microorganisms were isolated from the soil of a ginseng field using Esculin-R2A agar. Among these isolates, the strain GH21 showed the strongest activities to convert ginsenoside Rb₁ and Rg₁ to minor ginsenosides compound-K and F₁, respectively. Ginsenosides Rb₁ and Rg₁ bioconversion rates were 74.2 and 89.3%, respectively. Meanwhile, the results demonstrated that the ginsenoside Rg₁ could change the biotransformation pathway of ginsenoside Rb₁ by inhibiting the formation of the intermediate metabolite gypenoside-XVII. GH21 was identified as a Cladosporium cladosporioides species based on the internal transcribed spacers (ITS) ITS1-5.8S-ITS2 rRNA gene sequences constructed phylogenetic trees.     

An endophytic bacterium isolated from Panax ginseng C.A. Meyer enhances growth,reduces morbidity,and stimulates ginsenoside biosynthesis

Ginseng (Panax ginseng C.A. Meyer) is known for its therapeutically useful ginsenosides that have anticancer and other pharmacological effects. However, its low levels in plants and the high costs of chemical synthesis make ginsenosides commercially non-viable; as such, strategies for increasing ginsenoside yield are of great interest. The present study reports the isolation of eight novel endophytic bacteria from ginseng leaves, the highest ginsenoside concentration of microbial transformed strain was identified as Paenibacillus polymyxa. Inoculation of ginseng plants with P. polymyxa by foliar application combined with irrigation enhanced plant growth parameters, reduced morbidity, and increased plant concentration of the ginsenosides (Rg₁, Re, Rf, Rb₁, Rg₂, Rb₂, Rb₃, and Rd) in field experiments. These results indicate that P. polymyxa isolated from ginseng is a beneficial endophytic bacterium with biocontrol properties that can enhance the yield and quality of this medicinal plant.     

Improvement of ginsenoside production by <Emphasis Type="Italic">Panax ginseng</Emphasis> adventitious roots induced by γ-irradiation

In order to evaluate effects of γ-rays on adventitious root formation and ginsenoside production, embryogenic calli induced from cotyledon explants of Panax ginseng C.A. Meyer were treated with γ-rays of 0, 10, 30, 50, 70, and 100 Gy. The highest frequency of adventitious root formation of 75 % occurred at γ-irradiation of 30 Gy, which is considered adequate dosage for selecting mutant cell lines. Five mutated adventitious roots (MAR)3-lines out of the propagation of 142 adventitious root lines treated with 30 Gy were selected based a 100-fold increase in proliferation rate compared to control adventitious roots (CAR) and content of the seven major ginsenosides (Rb₁, Rb₂, R_c, R_d, R_e, R_f, and Rg₁) was determined. In the CAR and four of the MAR3-lines (except for MAR3-109), the Rb/Rg ratio was greater than 1.0, thereby indicating altered ginsenoside composition in these root lines. The HPLC analysis of the MAR3-13 and MAR3-26 lines confirmed different ginsenoside profiles, including the three unidentified ginsenoside candidates, Gm₁, Gm₂, and Gm₃. The ginsenosides of the MAR3-13 and MAR3-26 lines showed high hydroxyl and superoxide radical scavenging activities.     

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb₁ is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb₁ to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb₁ had been consecutively converted to ginsenoside Rd (1), F₂ (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb₁.     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.     

Quantitative Comparison of Ginsenosides and Polyacetylenes in Wild and Cultivated American Ginseng

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg₁/Rd ratio presented a significantly large difference between cultivated and type‐I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg₁+Re)/Rd, and the ratio of protopanaxatriol (PPT)‐type ginsenosides to protopanaxadiol (PPD)‐type ginsenosides showed a large difference between these two groups. On the other hand, type‐II wild samples were found to have high Rg₁/Rb₁ and Rg₁/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type‐I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type‐II wild American ginseng may have distinct pharmacological activities and therapeutic effects.     

Introgression of bread wheat chromatin into tall wheatgrass via somatic hybridization

Regenerates were obtained following somatic hybridization between tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum cv. Jinan177) protoplasts. Two lines (CU and XI) were self-fertile in the first (R₀) and subsequent (R₁ and R₂) generations. The phenotype of each R₁ population was uniform. All CU progeny were phenotypically similar to the tall wheatgrass parent, while XI progeny had thinner, smoother and softer leaves. Cytological analysis showed that more wheat chromatin was present in the hybrid callus than in the R₁ and R₂ plants, and that some intercalary translocations of wheat chromosome segments were retained in the R₂ generation. AFLP profiling confirmed the presence of wheat DNA in the introgression lines. Analysis of the high molecular weight glutenin subunit content of derived seed identified three novel subunits, not present in either the wheat or the tall wheatgrass parent. Microsatellite-based profiling of the chloroplast genome of the introgression lines suggested that only chloroplast sequences from the tall wheatgrass parent were present. The specifically inherited phenomena and possible application of these hybrids are discussed. Haifeng Cui and Zhiyong Yu were contributed equally to this article.     

A new ginsenosidase from Aspergillus strain hydrolyzing 20-O-multi-glycoside of PPD ginsenoside

A ginsenosidase specifically hydrolyzing multi-20-O-glycosides of protopanaxadiol type ginsenosides such as ginsenoside Rb₁, Rb₃, Rb₂ and Rc, named ginsenosidase type II, was isolated and purified from Aspergillus sp.g48p strain. The molecular weight of the enzyme was 60 kDa. Ginsenosidase type II was demonstrated to hydrolyze multi-20-O-glycoside of protopanaxadiol type ginsenoside Rb₁, Rb₃, Rb₂ and Rc; i.e. the ginsenosidase type II hydrolyzes 20-O-β-glucoside of the ginsenoside Rb₁, 20-O-β-xyloside of ginsenoside Rb₃, 20-O-α-arabinoside(p) of ginsenoside Rb₂ and α-arabinoside(f) of ginsenoside Rc to produce mainly ginsenoside Rd, and small amount of Rg₃. However, it did not hydrolyze 3-O-β-glucosides of ginsenoside Rb₁, Rb₃, Rb₂ and Rc which was different with the ginsenosidase type I previously reported, either did not hydrolyze the glycosides of protopanaxatriol type ginsenoside such as ginsenoside Re, Rf and Rg₁, showing significant difference from all previously described glycosidases.     

Ginsenosidases and the pathogenicity of Pythium irregulare

American ginseng (Panax quinquefolius L.) produces triterpenoid saponins, ginsenosides, that possess mild fungitoxic activity toward some common ginseng leaf pathogens. However, numerous oomycete root pathogens of ginseng, most notably Pythium irregulare Buisman, are able to partially deglycosylate 20 (S)-protopanaxadiol ginsenosides Rb1, Rd and gypenoside XVII via extracellular glycosidases, leading to a common product, ginsenoside F2. Conversion of the common 20 (S)-protopanaxadiols into F2 requires both β (1 → 6) and β (1 → 2) glucosidase activity. In the present study, the ability of nine distinct isolates of P. irregulare, as well as a P. ultimum Trow isolate and two isolates of Trichoderma hamatum (Bonord.) Bainier, to deglycosylate 20 (S)-protopanaxadiols, in vitro was examined. The pathogenicity of each isolate was also examined by scoring the severity of disease symptoms caused by each in separate inoculations of one- and two-year old ginseng seedlings. Disease severity was scored using a disease severity index, as well as by taking F_v/F_m measurements of leaves during a 14-day infection period. Based on these measurements, it was concluded that (1) the use of direct F_v/F_m measurements correlates strongly with observations of disease severity (R² = 0.79), and that (2) the pathogenicity of P. irregulare isolates correlates with their ability to deglycosylate ginsenosides (R² = 0.57). These results further support the hypothesis that the pathogenicity of P. irregulare on ginseng roots is dependent, in part, on the ability of this organism to deglycosylate ginsenosides.     

Highly selective microbial transformation of major ginsenoside Rb(1) to gypenoside LXXV by Esteya vermicola CNU120806

Aims

This study examined the biotransformation pathway of ginsenoside Rb₁ by the fungus Esteya vermicola CNU 120806.

Methods and Results

Ginsenosides Rb₁ and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb₁. The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and gypenoside LXXV. A mole yield of 95·4% gypenoside LXXV was obtained by enzymatic conversion (pH 5·0, temperature 50°C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49·6%) proved a consecutive hydrolyses occurred at the C‐3 position of ginsenoside Rb₁.

Conclusions

Strain CNU 120806 showed a high degree of specific β‐glucosidase activity to convert ginsenosides Rb₁ and Rd to gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50°C and pH 5·0. Compared with its activity against pNPG (100%), the β‐glucosidase exhibited quite lower level of activity against other aryl‐glycosides. Enzymatic hydrolysate, gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C‐3 carbon of ginsenoside Rb₁ and Rd, giving the pathway: ginsenoside Rb₁→ gypenoside XVII → gypenoside LXXV; ginsenoside Rd→F₂→Compound K, but did not hydrolyse the 20‐C, β‐(1‐6)‐glucoside of ginsenoside Rb₁ and Rd.

Significance and Impact of the Study

The results showed an important practical application on the preparation of gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.     

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.     

Characteristics of carrot plant regenerated from two cell line selected as either ionic-Al tolerant cell or Al-phosphate utilizing cells

Plantlets of carrot (Daucus carota L.) were regenerated from two types of cell lines. One type was selected as ionic-Al tolerant (IAT) cells, while the second type featured Al-phosphate utilizing cells (IPG). Their tolerance characteristics were investigated. The plantlets from IAT were directly regenerated, whereas those from IPG were regenerated after somatic hybridization with wild-type cells previously inactivated with iodacetamide, because the IPG cells had completely lost the ability to regenerate naturally.The sexual progeny of IAT showed Al-tolerant properties, established by testing their root elongation in the presence of 500 µM Al ions. Most of the calli obtained from the somatic hybrids grew more rapidly than the wild-type cells when Al-phosphate was used as a sole source of phosphorus. Thus, we obtained two types of carrot plantlets, regenerated from IAT and IPG. Both possessed the tolerant characteristics as observed with the stress-selected cells.     

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

Summary Protoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).     

Biotransformation of Ginsenosides (Rb1, Rb2, Rb3, Rc) in Human Intestinal Bacteria and Its Effect on Intestinal Flora

Ginsenosides, including Rb₁, Rb₂, Rb₃ and Rc, belong to protopanaxadiol-type saponins in Panax ginseng C. A. Mey. Their contents are high in P. ginseng. They could inhibit oxidant stress, enhance immunity, lower blood sugar, resist tumor cells and facilitate other physiological activities. This study aimed to explore the interaction between ginsenosides Rb₁, Rb₂, Rb₃ and Rc and the intestinal flora of healthy people. It also sought to analyse the biotransformation products and pathways of these ginsenosides in in-vitro human intestinal bacteria and their effects on the diversity of human intestinal flora. Human intestinal bacteria were incubated with ginsenosides Rb₁, Rb₂, Rb₃ and Rc at 37 °C under anaerobic conditions. Samples were taken at different timepoints. The transformed products were identified by rapid high-resolution liquid chromatography-quadrupole time-of-flight mass spectrometry. After 48 h of transformation, the transformed product of ginsenosides Rb₁, Rb₂, Rb₃ and Rc was ginsenoside compound K. The transformation rates were 83.5 %, 88.7 %, 85.6 %, and 84.2 %. 16S rRNA sequencing technology was applied to the bioinformatic analysis of faecal samples incubated for 48 h. Relative to the blank control, the relative abundance of Firmicutes and Proteobacteria significantly increased at the phylum level. Moreover, the relative abundance of Bacteroidetes significantly decreased in ginsenosides Rb₁, Rb₂, Rb₃ and Rc. At the genus level, the relative abundance of Escherichia significantly increased, whereas that of Dorea, Prevotella and Megasphaera significantly decreased in all groups. These results showed that Rb₁, Rb₂, Rb₃ and Rc could improve the structure and diversity of human intestinal flora and balance the metabolic process.     

Headspace Solid-Phase Micro-Extraction Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS)-Based Untargeted Metabolomics Analysis for Comparing the Volatile Components from 12 <i>Panax</i> Herbal Medicines

Quality control of ginseng currently is mainly based on ginsenoside analysis, but rarely focuses on the volatile organic components. In the current work, an untargeted metabolomics approach, by headspace solid-phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS), was elaborated and further employed to holistically compare the compositional difference of the volatile components simultaneously from 12 Panax herbal medicines, which included P. ginseng (PG), P. quinquefolius (PQ), P. notoginseng (PN), red ginseng (PGR), P. ginseng leaf (PGL), P. quinquefolius leaf (PQL), P. notoginseng leaf (PNL), P. ginseng flower (PGF), P. quinquefolius flower (PQF), P. notoginseng flower (PNF), P. japonicus (PJ), and P. japonicus var. major (PJvm). Chromatographic separation was performed on an HP-5MS elastic quartz capillary column using helium as the carrier gas, enabling good resolution within 1 h. We were able to characterize totally 259 volatile compounds, including 82 terpenes (T), 46 alcohols (Alc), 29 ketones (K), 25 aldehydes (Ald), 21 esters (E), and the others. By analyzing 90 batches of ginseng samples based on the untargeted metabolomics workflows, 236 differential ions were unveiled, and accordingly 36 differential volatile components were discovered. It is the first report that simultaneously compares the compositional difference of volatile components among 12 Panax herbal medicines, and useful information is provided for the quality control of ginseng aside from the well-known ginsenosides.     

Fungal sensitivity to and enzymatic deglycosylation of ginsenosides

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F₂. By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.     

Cell culture system versus adventitious root culture system in Asian and American ginseng: a collation

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g^?1 DW, 12.88 mg·L^?1, respectively) than that of CS cell line (9.50 mg·g^?1 DW, 2.39 mg·L^?1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.