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.     

Growth and biosynthetic characteristics of ginseng (Panax japonicus var. repens) deep-tank cell culture in bioreactors

The aim of the work was to study the growth characteristics of cultured cells of Panax japonicus var. repens, an endemic plant of the Primorski Krai of Russia, grown in laboratory bioreactors and to determine the content of basic ginsenosides under these conditions. An increase of the inoculum size of the culture produced higher biomass accumulation and economic coefficient but slightly reduced the specific growth rate. An increase in the auxin concentration in a medium by adding 2,4-D practically did not affect growth characteristics of the culture but significantly reduced the size of cell aggregates. In all treatments tested, all major ginsenosides (Rb₁, Rc, Rb₂, Rd, Rf, Rg₁, and Re) were found in the culture. The total ginsenoside content was 2–3% per biomass dry weight. Meantime, ginsenosides of the Rg-series with protopanaxatriol as aglycone prevailed (70% of the total ginsenoside content). The culture conditions considerably affected the ratio of individual ginsenosides. In 2,4-D-containing medium, the preferential synthesis of Re ginsenoside was observed while both Rg₁ and Re were synthesized in other treatments.     

The mechanism of acid-catalyzed conversion of ginsenoside Rf and two new 25-hydroxylated ginsenosides

Ginsenoside Rf is known to have higher chemical stability than other ginsenosides and until lately, the constituents in which it would convert were not known. Only in recent times, it was found that ginsenoside Rf converted to (20E)-Rg₉, (20Z)-Rg₉, Rg₁₀, and 20(R)-Rf. During my continued studies to update the chemical profile of red ginseng, two new ginsenosides converted from ginsenoside Rf, 25-hydroxylated ginsenosides, were discovered. These two new converted ginsenosides, namely (20E),25(OH)-ginsenoside Rg₉ (1), and (20Z),25(OH)-ginsenoside Rg₉ (2), together with ginsenosides (20E)-Rg₉ (3), (20Z)-Rg₉ (4), Rg₁₀ (5), and 20(R)-Rf (6) were isolated from a reaction mixture of ginsenoside Rf in an acid-catalyzed reaction. Their chemical structures (1 and 2) were elucidated by NMR and Mass spectral methods. Compounds 1 and 2 were presumably generated by hydration of (20E)-, and (20Z)-ginsenoside Rg₉. The presence of these six converted ginsenosides was confirmed by UPLC/TOF-MS method in red ginseng. On the basis of these results, I deduced the overall conversion mechanism of ginsenoside Rf and evaluated the significance of ginsenoside Rf as a characteristic mark substance of Panax ginseng.     

Autotoxic Ginsenosides in the Rhizosphere Contribute to the Replant Failure of Panax notoginseng

MethodsThe autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS.ResultsThe seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including R₁, Rg₁, Re, Rb₁, Rb₃, Rg₂, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg₁, Re, Rg₂, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R₁, Rg₁, Re, Rg₂, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL.ConclusionOur results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth.     

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.     

Comparison of ginsenoside composition in native roots and cultured callus cells of Panax quinquefolium L.

In order to compare the ginsenoside composition in native Panax quinquefolium and in suspension cultured cells derived from root callus, HPLC–ESI-MSⁿ analysis was performed. Under the present HPLC–ESI-MSⁿ conditions, ten ginsenosides from native root were acquired in the positive and negative ion modes, namely Rg₁, Re, Ro, malonyl-Rb₁, Rf, Rb₁, Rc, Rb₂, Rb₃ and Rd. Only four ginsenosides (Rg1, Re, Rf and Rb1) were identified from callus cells. Radical scavenging activity of P. quinquefolium callus cells with 250 mg l^?1 methanolic extract on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was 55.72 %, while only 6.31 % DPPH inhibition was obtained in native root.     

High Density Cultivation of Panax notoginseng Cell Cultures with Methyl Jasmonate Elicitation in a Centrifugal Impeller Bioreactor

True ginseng roots contain “active compounds” called ginsenosides. The enhanced production of useful bioactive ginsenosides by high‐density cell cultures of Panax notoginseng in a self‐developed centrifugal impeller bioreactor (CIB) was achieved by adding methyl jasmonic acid (MJA) during cultivation. The production of the major, individual ginsenosides Rg₁, Re and Rb₁ was significantly enhanced in both 3‐L and 30‐L CIBs. The production titer of Rg₁, Re and Rb₁ ginsenosides in the 30‐L CIB was improved from 42 ± 8, 42 ± 9 and 41 ± 6 mg/L without MJA elicitation, to 104 ± 6, 71 ± 5 and 95 ± 6 mg/L with MJA elicitation, respectively. The ratio of Rb/Rg was slightly improved by MJA treatment in a 3‐L CIB but no apparent difference was observed in a 30‐L CIB. This work is useful for the understanding of the effects of large‐scale production on the individual ginseng saponins produced by plant cell cultures     

Ginsenoside F1 production from ginsenoside Rg1 by a purified β-glucosidase from Fusarium moniliforme var. subglutinans

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F₁ from ginsenoside Rg₁. The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg⁻¹. It hydrolysed glucose-linked ginsenosides Rb₁, Rd and Rg₁ but not for other monosaccharide-linked ginsenosides, Rb₂, Rc, R₁, and Re. Under the optimum conditions of pH 6.0, 50°C, 30 U l⁻¹ of enzyme, and 5 mg Rg₁ ml⁻¹, 4 mg F₁ ml⁻¹ was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l⁻¹ h⁻¹. This represents the highest productivity and conversion yield of F₁ yet reported.     

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.     

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.     

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.     

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.     

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.

     

Lactobacillus ginsenosidimutans sp. nov., isolated from kimchi with the ability to transform ginsenosides

Biotransformation of ginsenosides was examined using lactic acid bacteria isolated from several kinds of kimchi. A Gram-positive, facultatively anaerobic, non-motile, non-spore-forming, and rod-shaped lactic acid bacterial strain, designated EMML 3041^T, was determined to have ginsenoside-converting activity and its taxonomic position was investigated using a polyphasic approach. Strain EMML 3041^T displayed β-glucosidase activity that was responsible for its ability to transform ginsenoside Rb₁ (one of the dominant active components of ginseng) to F₂ via gypenoside XVII, ginsenoside Rb₂ to compound Y via compound O, ginsenoside Rc to compound Mc via compound Mc₁, and ginsenoside Rd to ginsenoside F₂. On the basis of the 16S rRNA gene sequence similarity, strain EMML 3041^T was shown to belong to the genus Lactobacillus and is closely related to Lactobacillus versmoldensis KU-3^T (98.3 % sequence similarity). Polyphasic taxonomy study confirmed that the strain EMML 3041^T represents a novel species, for which the name Lactobacillus ginsenosidimutans sp. nov. is proposed, with EMML 3041^T (=KACC 14527^T = JCM 16719^T) as the type strain.     

Ginsenoside Rb1 in asymmetric somatic hybrid calli of Daucus carota with Panax quinquefolius

American ginseng (Panax quinquefolius L.) is one of the most valuable herbs in the world. Its major active components are ginsenosides. In order to produce ginsenoside heterogeneously, somatic hybridization, a novel approach for genetic introgression, was employed in this study. Protoplasts derived from respective calli of carrot (Daucus carota var. sativus Hoffm.) and American ginseng (P. quinquefolius L.) were used as the fusion partners. Hybrid calli derived from single cell lines containing chromatin of American ginseng were confirmed by the analyses of isozyme, Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH). High performance liquid chromatography (HPLC) results showed that the ginseng monomer Rb₁ was synthesized in seven of the hybrid calli identified as well as in the parent American ginseng calli but not in the parent carrot calli. Results indicated that hybrid introgression lines could produce ginsenoside Rb₁ and the ginsenoside Rb₁ biosynthesis pathway has been introgressed into carrot cells via somatic hybridization. From the point of biosafety view concerning the consumer acceptance, the potential predominance to produce ginsenosides with somatic hybridization other than with genetic transformation is discussed. Lu Han and Chuanen Zhou contributed equally to this work.     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides

This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028^T in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb₁ and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F₂, and Rh₂(S) with various pathways. BglAm can effectively transform the ginsenoside Rb₁ to gypenoside XVII and Rd to F₂; the K _m values of Rb₁ and Rd were 0.69?±?0.06 and 0.45?±?0.02 mM, respectively, and the V _max values were 16.13?±?0.29 and 51.56?±?1.35 μmol min^?1 mg^?1 of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg₁ into Rg₂(S) and Rh₁(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.     

Production of saponins from <Emphasis Type="Italic">Panax ginseng</Emphasis> suspension and adventitious root cultures

Biomass growth and ginsenoside production in cell suspension and adventitious roots of Panax ginseng C.A. Meyer cultures cultivated both in Erlenmayer flasks and a 3 dm³ bioreactor were studied. The maximum content of ginsenosides was found in the suspension culture cultivated in the bioreactor (4.34 % dry mass), however the saponin content was limited to two major ginsenosides, Rb₁ and Rg₁. The production of ginsenosides in adventitious roots was lower (1.45 or 1.72 % dry mass), nevertheless, the full range of ginsenosides was detected.This work was supported by 521/02/P064, COST 843.10, ME671 and Z4 055 905 projects.     

Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures

We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures. Carbachol stimulated formation of [³H]inositol phosphates ([³H]InsPs) by 3.3-fold over basal level in [³H]inositol-prelabeled cells. Pretreatment of GTS inhibited formation of [³H]InsPs evoked by carbachol by 70%–90%. Addition of GTS alone had no effect on the basal formation of [³H]InsPs. The inhibitory effect of the GTS on carbachol-stimulated formation of [³H]InsPs was dose- and time-dependent. IC₅₀ was 6.0 ± 2.8 g/ml. We also examined the effect of GTS on [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol. Although GTS had no effect on the basal [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation, pretreatment of GTS inhibited [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol, respectively. Addition of individual ginsenosides such as ginsenoside Rb₁, Rc, Rd, Re, or Rg₂ had no effect on the basal formation of [³H]InsPs, whereas pretreatment of ginsenoside Rb₂, Rc, Rd, Re, Rf, Rg₁ or Rg₂ inhibited formation of [³H]InsPs evoked by carbachol by 79%–89%. The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of [³H]InsPs in cortical neurons could be one pharmacological action of Panax ginseng.