Ginsenoside Accumulation Research Articles

Promotion of ginsenosides production in a co-cultivation system of Panax ginseng adventitious roots and immobilized Aspergillus niger

Biotic elicitor is an important biotechnology strategy to promote metabolite production in adventitious root culture. In this study, Panax ginseng adventitious roots (PGARs) were co-cultured with sterilized mycelium powders (SMPAN), immobilized mycelium (IMAN), and immobilized spores of Aspergillus niger (ISAN) respectively for the enhancement of ginsenosides accumulation. In this co-cultivation system, ISAN was selected as the best inducer. The maximum ginsenosides content (26.43 ± 0.49 mg/g) were obtained in the ISAN spore concentration of 102 spores/mL, the volume ratio of 4%, and co-culture time of 24 h, which was 3.09 times higher than non-treated control (8.55 ± 0.78 mg/g). Besides, the lactic acid secreted by A. niger was identified as the main component inducing ginsenosides accumulation. Moreover, this study revealed that lactic acid could trigger the instantaneous accumulation of signaling molecules and enhance the expression of transcription factors and defense genes in PGARs, thus resulting in the up-regulated expression of functional genes involved in ginsenosides biosynthetic pathway. Therefore, co-culture of ISAN and PGARs is a very promising method to increase ginsenosides production. It provides a new way to promote the accumulation of secondary metabolites in plants.

Cold-induced ginsenosides accumulation is associated with the alteration in DNA methylation and relative gene expression in perennial American ginseng (Panax quinquefolius L.) along with its plant growth and development process

BackgroundGinsenosides accumulation responses to temperature are critical to quality formation in cold-dependent American ginseng. However, the studies on cold requirement mechanism relevant to ginsenosides have been limited in this species. MethodsTwo experiments were carried out: one was a multivariate linear regression analysis between the ginsenosides accumulation and the environmental conditions of American ginseng from different sites of China and the other was a synchronous determination of ginsenosides accumulation, overall DNA methylation, and relative gene expression in different tissues during different developmental stages of American ginseng after experiencing different cold exposure duration treatments. ResultsResults showed that the variation of the contents as well as the yields of total and individual ginsenosides Rg1, Re, and Rb1 in the roots were closely associated with environmental temperature conditions which implied that the cold environment plays a decisive role in the ginsenoside accumulation of American ginseng. Further results showed that there is a cyclically reversible dynamism between methylation and demethylation of DNA in the perennial American ginseng in response to temperature seasonality. And sufficient cold exposure duration in winter caused sufficient DNA demethylation in tender leaves in early spring and then accompanied the high expression of flowering gene PqFT in flowering stages and ginsenosides biosynthesis gene PqDDS in green berry stages successively, and finally, maximum ginsenosides accumulation occurred in the roots of American ginseng. ConclusionWe, therefore, hypothesized that cold-induced DNA methylation changes might regulate relative gene expression involving both plant development and plant secondary metabolites in such cold-dependent perennial plant species.

Preliminary study on response and its mechanism of ginsenoside biosynthesis in Panax ginseng to water regulation

The study is aimed to explore the effect of soil moisture content on ginsenoside biosynthesis and explain its mechanism from the perspectives of antioxidant enzyme system and gene expression of key enzymes in the pathway of ginsenoside synthesis. In the study,two years old Panax ginseng was used as the experimental material and three moisture gradient,40% of saturated water content( W1),60%( W2),80%( W3) were set up. The content of 11 monomeric saponins were determined by HPLC. With GAPDH as a reference gene,six key enzymes( HMGR,SS,β-AS,CYP716 A47,CYP716 A52 v2,CYP716 A53 v2) in ginseng saponin synthesis pathway expression were analyzed by fluorescent quantitative PCR and the activities of superoxide dismutase( SOD),peroxidase( POD),catalase( CAT) activity and MDA content were also determined. With the increase of soil water,the content of ginseng saponin and biomass showed an increasing trend. PPD( Rb1,Rc,Rb2,Rd,Rh2,Rb3,Rg3),PPT( Rg1,Re,Rf) ginsenoside,Ro and total ginsenoside reached the maximum value on August 30,were 9.92,5.48,0.63 mg·g-1,respectively. During the whole regulation period,the antioxidant activity of W3 was greater than that of W1,and the MDA content was less than that of W1. At W3,expression levels of β-AS,CYP716 A47 and CYP716 A53 v2 showed an increasing trend,while HMGR and SS genes showed relatively stable expression levels under various water conditions. According to the correlation analysis,HMGR and SS genes in the W3 treatment group were significantly positively correlated with PPD,PPT ginsenoside and Ro,CYP716 A52 v2 gene was significantly positively correlated with Ro,and CYP716 A47 gene was significantly positively correlated with PPD ginsenoside. There was a significant positive correlation between β-AS gene and PPD ginsenoside in W1 and W2 treatment. Therefore,W3 is the optimum moisture content,ginseng total saponins and monomer saponin content is the highest,the gene closely correlation with content of saponins and more conducive to the accumulation of ginsenosides.

Quality evaluation of Panax ginseng adventitious roots based on ginsenoside constituents, functional genes, and ferric-reducing antioxidant power.

In the study, six adventitious root lines of Panax ginseng have been successfully established. HPLC-ESI-MS analysis showed that 20 ginsenosides were identified in root lines, notoginsenoside Fa and notoginsenoside R2 were not found in AR lines. In AR lines, the highest accumulation of total ginsenosides was obtained in five-year main AR (24.87mg/g). Principal component analysis classified root lines into three groups. Five-year ginseng was mostly similar with five-year main AR, five-year rootlet AR, and four-year rootlet AR in ginsenosides composition of group 1. Besides, gene expressions were consistent with the production of total ginsenosides, and correlation analysis revealed that total ginsenosides biosynthesis was significantly positively correlated with the gene expression of dammarenediol synthase. Five-year rootlet AR showed the highest activity on ferric-reducing antioxidant power test among samples. It provides a scientific evidence for the further exploitation and large-scale production of P. ginseng. PRACTICAL APPLICATIONS: This study provides valuable information for the commercial scale culture of ginseng adventitious roots. This report combines morphology, ginsenoside composition and content, gene expression, and ferric-reducing antioxidant power test to evaluate the quality of P. ginseng adventitious root, and combined with principal component analysis to screen out the high yield and stable ginseng adventitious roots. It would be profitable to use adventitious root culture of P. ginseng instead of field cultivation.

Steaming combined with biochar application eliminates negative plant-soil feedback for sanqi cultivation

Eliminating negative plant-soil feedback (NPSF) in agricultural production systems is the key step in maintaining soil health and sustainable crop production. Sanqi (Panax notoginseng) has been severely hampered by NPSF due to the build-up of soilborne pathogens and the accumulation of autotoxic ginsenosides. Steaming on soil followed by adding biochar was investigated. Greenhouse experiments demonstrated that consecutively cultivated soil treated with steaming (90 °C for 15 min) significantly increased seed germination rates and seedling survival rates of sanqi, but biochar amendment had no effects. The combination of steaming with biochar amendments (8 g L−1) was more effective on seed germination and seedling survival rates as well as increasing the biomass of sanqi than applying steaming alone. Steaming slightly degraded the content of autotoxic ginsenosides in the soil and completely eliminated the population of soilborne pathogens, including Ilyonectria spp. and Fusarium spp., in consecutively cultivated soil, and enhanced the release of nutritive substances into soil. Biochar showed a high adsorption capacity for autotoxic ginsenosides, and increased nutrient supplementation. Steaming plus biochar could effectively eliminate the negative effects of soilborne pathogens and autotoxicants to P. notoginseng. Four years of field studies further confirmed that consecutively cultivated soil could be successfully replanted after steaming plus biochar application. Thus, steaming plus biochar amendment was a promising practice for enhancing soil quality and health.

Effect of temperature on morphology, ginsenosides biosynthesis, functional genes, and transcriptional factors expression in Panax ginseng adventitious roots.

This study researched the effect of temperature on growth and ginsenosides accumulation in adventitious root cultures of Panax ginseng. Results showed that the ginseng adventitious roots growth and differentiation ability could be affected faced with different incubation temperatures (15, 20, 25, and 30°C for 35days). Besides, the research also demonstrated that low-temperature stimulation could promote the accumulation of ginsenosides and the content of total ginsenosides increased by 2.53 times at 10°C-7d (10°C for 7days and then transferred to 25°C for 28days) compared with that at 25°C. Moreover, the transcriptional levels of functional genes and PgWRKYs were analyzed by this study and the correlation analysis showed that GPS, SS, CYP716A47, CYP716A53v2, UGT74AE2, UGT94Q2, PgWRKY1, PgWRKY3, and PgWRKY8 were significantly correlated with total ginsenosides content. Furthermore, HPLC-ESI-MSn analyzed that Malonyl-Rb1 only existed in 10°C-7d group. PRACTICAL APPLICATIONS: The survey showed that after a certain time of stimulating P. ginseng adventitious roots at low temperature, the accumulation of ginsenosides could be enhanced as their expression of related genes were regulated. It provides a theoretical foundation for the mass production of ginsenosides by controlling the temperature conditions of P. ginseng adventitious roots.

The Effects of Environmental Factors on Ginsenoside Biosynthetic Enzyme Gene Expression and Saponin Abundance

Panax ginseng C.A. Meyer is one of the most important medicinal plants in Northeast China, and ginsenosides are the main active ingredients found in medicinal ginseng. The biosynthesis of ginsenosides is regulated by environmental factors and the expression of key enzyme genes. Therefore, in this experiment, ginseng in the leaf opened stage, the green fruit stage, the red fruit stage, and the root growth stage was used as the test material, and nine individual ginsenosides and total saponins (the sum of the individual saponins) were detected by HPLC (High Performance Liquid Chromatography). There was a trend of synergistic increase and decrease, and saponin accumulation and transfer in different tissues. The expression of key enzyme genes in nine synthetic pathways was detected by real-time PCR, and the correlation between saponin content, gene expression, and ecological factors was analyzed. Correlation analysis showed that in root tissue, PAR (Photosynthetically Active Radiation) and soil water potential had a greater impact on ginsenoside accumulation, while in leaf tissue, temperature and relative humidity had a greater impact on ginsenoside accumulation. The results provide a theoretical basis for elucidating the relationship between ecological factors and genetic factors and their impact on the quality of medicinal materials. The results also have guiding significance for realizing the quality of medicinal materials.

Salicylic acid and ultrasonic stress modulated gene expression and ginsenoside production in differentially affected Panax quinquefolius (L.) and Panax sikkimensis (Ban.) cell suspensions

Biomass and in vitro ginsenoside accumulation in cell suspensions of Panax quinquefolius (L.) and P. sikkimensis (Ban.) are differentially affected, under influence of salicylic acid (SA; 100 and 200 µM) and ultrasonic stress (US; 120 W US power, 15 s). SA addition to P. quinquefolius, was observed to lead to decline in biomass accumulation; however SA100 treatment for 5 days led to a 2.6-fold increase in ginsenoside production and Rg3 induction and exudation (6.4 mg/L). Marginally declined growth and ginsenoside productivity was observed on US exposure (% BI or biomass increment = 150.2, ginsenoside = 24.9 mg/L) as compared to unchallenged cultures (% BI = 157.5, ginsenoside = 27.2 mg/L). Co-application of US to SA100 and SA200 treatments for 5 days, although had no significant effect on cell biomass, however led to a further decline in ginsenoside productivity (SA100 + US = 48.6 mg/L, SA200 + US = 27.9 mg/L), when compared to cultures treated only with SA (SA100 = 70.5 mg/L, SA200 = 39.4 mg/L). On the other hand, addition of SA100 and SA200 to P. sikkimensis for 1 week led to a sharp decline in biomass and ginsenoside production, when compared to control cultures. Interestingly, growth and ginsenoside productivity was significantly improved upon co-application of US. US exposure was probably “boosting” mechanism of SA action (SA100 + US = %BI = 124.3, ginsenoside = 57.7 mg/L, SA200 + US = % BI = 135.6, ginsenoside = 102.17 mg/L), when compared to cultures treated with only SA (SA100 = % BI = 96.6, ginsenoside = 19.6 mg/L, SA200 = % BI 103.4, ginsenoside = 36.3 mg/L). In brief, SA100 was the best treatment for maximum ginsenoside productivity specially ginsenoside Rg3 from P. quinquefolius, whereas, SA200 + US was observed to be optimal for P. sikkimensis cell suspensions.

Ginsenoside accumulation profiles in long- and short-term cell suspension and adventitious root cultures in Panax ginseng

In this study, we compared two in vitro culture systems, dedifferentiated cell suspension culture and differentiated adventitious root culture, using inoculum from long-term (20-year culture period) and short-term (1-year culture period) cultures, for the production of ginsenosides in wild ginseng (Panax ginseng). Increases in biomass and ginsenoside content were monitored in a 3-L bioreactor. The biomass in short-term cell suspension and adventitious root cultures increased at a faster rate than that in the long-term cultures, reaching a peak after 4 and 8 weeks of culture, respectively. In long term cultured materials, the biomass of cell suspension and adventitious root cultures decreased by 1.67- and 1.52-fold, respectively. Although the amount of ginsenosides in cell suspension and adventitious root cultures varied during the culture periods, the total ginsenoside content remained constant. These data suggest that both culture systems are advantageous for the production of different ginsenosides for pharmaceutical purposes.

Enhanced expression of ginsenoside biosynthetic genes and in vitro ginsenoside production in elicited Panax sikkimensis (Ban) cell suspensions.

Dual metabolite, i.e., ginsenoside and anthocyanin, co-accumulating cell suspensions of Panax sikkimensis were subjected to elicitation with culture filtrates of Serratia marcescens (SD 21), Bacillus subtilis (FL11), Trichoderma atroviridae (TA), and T. harzianum (TH) at 1.25% and 2.5% v/v for 1- and 3-week duration. The fungal-derived elicitors (TA and TH) did not significantly affect biomass accumulation; however, bacterial elicitors (SD 21 and FL11), especially SD 21, led to comparable loss in biomass growth. In terms of ginsenoside content, differential responses were observed. A maximum of 3.2-fold increase (222.2mg/L) in total ginsenoside content was observed with the use of 2.5% v/v TH culture filtrate for 1week. Similar ginsenoside accumulation was observed with the use of 1-week treatment with 2.5% v/v SD 21 culture filtrate (189.3mg/L) with a 10-fold increase in intracellular Rg2 biosynthesis (31mg/L). Real-time PCR analysis of key ginsenoside biosynthesis genes, i.e., FPS, SQS, DDS, PPDS, and PPTS, revealed prominent upregulation of particularly PPTS expression (20-23-fold), accounting for the observed enhancement in protopanaxatriol ginsenosides. However, none of the elicitors led to successful enhancement in in vitro anthocyanin accumulation as compared to control values.

Influence of exogenous ginsenosides on new forest soil microbial communities

Ginsenosides are the main active ingredient and allelochemicals of Panax ginseng, and they play an important role in ginseng growth and in ecological adaptation. To study the influence of ginsenosides on soil microbial communities, the method of given exogenous total ginsenosides of different concentrations were used to study the influence of ginsenosides on new forest soil microbial community, evaluate the change of metabolic activity of microbial community and investigate the ecological effect of ginsenosides on soil microbial community. Results showed that, exogenous total ginsenosides promoted metabolic activity of microbial community in new forest soil at different concentrations compared with the control after 10 d and 40 d treatment. After 10 d,except for the Evenness index, all of the other indices indicated that the functional diversity of the soil microbial community in the new forest firstly increased then decreased with increase of the total ginsenosides concentration. The Substrate richness for 0.01 g•L⁻¹ soil treatment was significantly different from that of the control. After 20 d, 30 d and 40 d, except for the Evenness index, all of the other indices indicated that the functional diversity of the soil microbial community in the new forest increased with total ginsenosides. These results suggested that ginsenosids can change soil microbial community and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of ginseng with accumulation of ginsenosides in the soil.

Endophytic Bacteria Isolated from Panax ginseng Improves Ginsenoside Accumulation in Adventitious Ginseng Root Culture.

Ginsenoside is the most important secondary metabolite of ginseng. Natural sources of wild ginseng have been overexploited. Although root culture could reduce the length of the growth cycle of ginseng, the number of ginsenosides is fewer and their contents are lower in adventitious roots of ginseng than that in ginseng cultivated in the field. In this study, we investigated the effects of endophytic bacterial elicitors on biomass and ginsenoside production in adventitious roots cultures of Panax ginseng. Endophyte LB 5-3 as an elicitor could increase biomass and ginsenoside accumulation in ginseng adventitious root culture. After 6 days elicitation with a 10.0 mL of strain LB 5-3, the content of total ginsenoside was 2.026 mg g−1 which was four times more than that in unchallenged roots. The combination of methyl jasmonate and strain LB 5-3 had a negative effect on ginseng adventitious root growth and ginsenoside production. The genomic DNA of strain LB 5-3 was sequenced, and was found to be most closely related to Bacillus altitudinis (KX230132.1). The challenged ginseng adventitious root extracts exerted inhibitory effect against the HepG2 cells, which IC50 value was 0.94 mg mL−1.

GC-MS Metabolomic Analysis to Reveal the Metabolites and Biological Pathways Involved in the Developmental Stages and Tissue Response of Panax ginseng.

Ginsenosides, the major compounds present in ginseng, are known to have numerous physiological and pharmacological effects. The physiological processes, enzymes and genes involved in ginsenoside synthesis in P. ginseng have been well characterized. However, relatively little information is known about the dynamic metabolic changes that occur during ginsenoside accumulation in ginseng. To explore this topic, we isolated metabolites from different tissues at different growth stages, and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 30, 16, 20, 36 and 31 metabolites were identified and involved in different developmental stages in leaf, stem, petiole, lateral root and main root, respectively. To investigate the contribution of tissue to the biosynthesis of ginsenosides, we examined the metabolic changes of leaf, stem, petiole, lateral root and main root during five development stages: 1-, 2-, 3-, 4- and 5-years. The score plots of partial least squares-discriminate analysis (PLS-DA) showed clear discrimination between growth stages and tissue samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in the same tissue at different growth stages indicated profound biochemical changes in several pathways, including carbohydrate metabolism and pentose phosphate metabolism, in addition, the tissues displayed significant variations in amino acid metabolism, sugar metabolism and energy metabolism. These results should facilitate further dissection of the metabolic flux regulation of ginsenoside accumulation in different developmental stages or different tissues of ginseng.

The integration of GC–MS and LC–MS to assay the metabolomics profiling in Panax ginseng and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary metabolites and ginsenosides accumulation

The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry. A complex continuous coordination of primary- and secondary-metabolic network was modulated by tissues and species factors during growth. The results showed that altogether 149 primary compounds and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P. ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed obvious compounds distinction among tissue-specific districts relative to species. To survey the dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides, we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the results indicated that high rates of C assimilation to C accumulation are closely associated with ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively. Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of the ginsenosides biosynthesis at the metabolite level.

Potential accumulation of protopanaxadiol-type ginsenosides in six-months toxicokinetic study of SHENMAI injection in dogs

SHENMAI injection (SMI), derived from famous Shen Mai San, is a herbal injection widely used in China. Ginsenosides are the major components of SMI. To monitor the exposure level of SMI during long-term treatment, a 6-month toxicokinetic experiment was performed. Twenty-four beagle dogs were dived into four groups (n = 6 in each group): a control group (0.9% NaCl solution) and three SMI groups (2, 6 or 3 mg/kg). The dogs were i.v. infused with vehicle or SMI daily for 180 d. Blood samples for analysis were collected at specific time points as follows: pre-dose (0 h); at 10, 30, and 60 min during infusion; and at 10, 30, 60, 90, 120, 240, and 300 min post-administration. Concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the plasma were determined simultaneously by liquid chromatography–tandem mass spectrometry. Non-compartmental parameters were further calculated and analyzed. Significant differences were found between the kinetic behavior of 20(S)-protopanaxadiol-type (PPD-type) and 20(S)-protopanaxatriol-type (PPT-type) ginsenosides. Increasing in the exposure level of PPD-type ginsenosides was observed in dogs during the experiment. Therefore, PPD-type ginsenosides are closely related to the immunity modulation effect of SMI. Increased PPD-type ginsenoside exposure level may present potential toxicity and induce drug-drug interaction risks during SMI administration. As such, PPD-type ginsenoside accumulation must be carefully monitored in future SMI research.

Profiling of ginsenosides in the two medicinal Panax herbs based on ultra-performance liquid chromatography-electrospray ionization-mass spectrometry.

As the king of herb plants, ginseng has been used for nearly 5000 years in medicines in Asia and recently in the West. Ginsenosides, the main active constituents in Panax herbs, have prominent immunoregulatory effects. Although extensively studied in the roots, ginsenosides have not been studied with regard to their profiles and natural variations in the leaf, stem, petiole, lateral root, and main roots during development or among species. In this study, a sensitive ultra-performance liquid chromatography-electrospray ionization–mass spectrometry method with a shorter chromatographic running time was developed and validated for simultaneous quantification of ten ginsenosides. Comparing ginsenoside contents in various parts during different developmental stages revealed part-specific accumulation of most ginsenosides. Further investigation indicated that Rg3 accumulated at significantly higher levels in the petiole of P. ginseng than in that of P. quinquefolius. The relative ratio of ginsenoside Rb2 to Rb1 appears to be a candidate metabolic marker for identifying the ginseng cultivar within a diverse collection of ginseng accessions. In addition, the PCA showed that aboveground parts differed significantly between species and can be considered as species-specific markers rather than roots. This comprehensive survey, providing reliable, affordable and adequate scientific evidence, could be used to differentiate two species and discriminate ginseng cultivar ages.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3427-3) contains supplementary material, which is available to authorized users.

Pathogenic fungal elicitors enhance ginsenoside biosynthesis of adventitious roots in Panax quinquefolius during bioreactor culture

Fungi have been widely used as biotic elicitors to promote metabolite production of plant cell, tissue, and organ cultures. Alternaria panax Whetz. and Cylindrocarpon destructans (Zinss) Scholten are the main pathogenic fungi in ginseng cultivation. The present study used extracts of A. panax and C. destructans as elicitors to simulate adventitious roots (ARs) of Panax quinquefolius L. for improvement of ginsenoside accumulation during AR bioreactor culture. To select a suitable elicitation method, the effects of treatment duration and concentration of both fungal extracts were investigated. After 30 d of AR bioreactor culture, the addition of A. panax and C. destructans extracts for 8 d to the culture medium produced higher ginsenoside content and productivity than the other treatment periods. During the elicitation of both fungal extracts, the peak contents of nitric oxide (NO), putrescine (Put), and ginsenoside were observed at 2, 4, and 8 d, respectively. Therefore, NO and Put could be upstream signals to regulate ginsenoside synthesis. The optimal elicitor concentration varied between extracts of A. panax and C. destructans. The maximum total ginsenoside content and productivity were observed with 4mg/L and 20mg/L of the A. panax and C. destructans extracts, respectively. The ginsenoside monomers with the most increase were Rg3, Rh2, and Re for the A. panax extract and Rg3, Rh2, and Rf for the C. destructans extract. In addition, the AR biomass was more inhibited by the C. destructans (20mg/L) extract, although its ginsenoside content (34.1mg/g DW) was higher than that with the A. panax (4mg/L) extract. The maximum ginsenoside production (276.0mg/L) was achieved with the A. panax (4mg/L) extract. Therefore, the A. panax extract was more suitable as an elicitor for ginsenoside production during AR bioreactor culture. The present study confirmed that treating 30-d-old P. quinquefolius ARs with 4mg/L A. panax extract for 8 d during bioreactor culture is the optimal elicitation method; these ARs can be applied to the commercial production of P. quinquefolius products.

Different chilling stresses stimulated the accumulation of different types of ginsenosides in Panax ginseng cells

Ginseng (Panax ginseng) is one of the most medically important plants in the world. Dammarane-type ginsenosides, which mainly include protopanaxatriol-type (PPT-type) and protopanaxadiol-type (PPD-type) ginsenosides, are the major pharmacologically relevant compounds that are produced by ginseng. Dammarenediol-II synthase (DDS) is the first committed enzyme in the ginsenoside biosynthetic pathway for dammarane-type ginsenosides, and PPD-type and PPT-type ginsenosides are catalyzed by protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), respectively. Ginseng cells are often used in stress studies. During their growth and development, ginseng plants are often exposed to cold stress. This study evaluated the effects of different chilling stresses on the accumulation of ginsenosides and the expressions of the DDS, PPDS and PPTS genes in ginseng cells. The results showed that continuous chilling (5 °C for 12 h) induced the PPT-type ginsenosides; whereas intermittent chilling (25 °C for 12 h and 5 °C for 12 h) stimulated the accumulation of PPD-type ginsenosides. The expression levels of DDS, PPDS and PPTS were clearly consistent with the accumulation pattern for PPT-type ginsenosides under continuous chilling stress or PPD-type ginsenosides under intermittent chilling stress, as was their order of involvement in the PPT-type or PPD-type biosynthetic pathway. These results indicate that different chilling treatments stimulated the accumulation of different types of ginsenosides, suggesting that cold stress may be one of the reasons for ginsenoside accumulation in ginseng cells.

SNPs of dammarenediol synthase gene were associated with the accumulation of ginsenosides in DAMAYA ginseng, a cultivar of Panax ginseng C. A. Mey.

ContextGinsenosides are regarded as the major active ingredients of ginseng, the dried taproot of Panax ginseng C. A. Mey. Our previous study has shown that several single nucleotide polymorphisms (SNPs) loci were identified in dammarenediol synthase (DS) gene, a key enzyme in the biosynthetic process of ginsenoside. ObjectiveThe current study aims to investigate the possible influence of SNPs on the content of ginsenosides in DAMAYA ginseng, a cultivar of P. ginseng. Materials and methodsWe selected a particular group of DAMAYA ginseng samples that were harvested in the same place of origin for SNP analysis, and then analyzed 18 ginsenosides in these samples with ultrahigh pressure liquid chromatography (UHPLC) coupled with time-of-flight mass spectrometer system (TOF/MS). ResultsWe found that these SNPs are associated with the accumulation of active ingredients ginsenosides Rf, Rg1 and F1. Moreover, OPLS-DA analysis implied that SNP loci of 279 might play a role in influencing the content level of ginsenosides. Further bioinformatics analysis showed that Leu83aa mutant might cause structural and functional changes of DS gene, which could in turn result in the content change of dammarane ginsenosides. Discussion and conclusionOur findings suggest that specific SNPs of DS gene might be associated with the accumulation of some ginsenosides in DAMAYA ginseng and thus can be used for varietal characterization, molecular identification, qualitative evaluation of ginseng-type medicinal materials, or gene engineering and ginseng breeding.

Proteomic Analyses Provide Novel Insights into Plant Growth and Ginsenoside Biosynthesis in Forest Cultivated Panax ginseng (F. Ginseng).

F. Ginseng (Panax ginseng) is planted in the forest to enhance the natural ginseng resources, which have an immense medicinal and economic value. The morphology of the cultivated plants becomes similar to that of wild growing ginseng (W. Ginseng) over the years. So far, there have been no studies highlighting the physiological or functional changes in F. Ginseng and its wild counterparts. In the present study, we used proteomic technologies (2DE and iTRAQ) coupled to mass spectrometry to compare W. Ginseng and F. Ginseng at various growth stages. Hierarchical cluster analysis based on protein abundance revealed that the protein expression profile of 25-year-old F. Ginseng was more like W. Ginseng than less 20-year-old F. Ginseng. We identified 192 differentially expressed protein spots in F. Ginseng. These protein spots increased with increase in growth years of F. Ginseng and were associated with proteins involved in energy metabolism, ginsenosides biosynthesis, and stress response. The mRNA, physiological, and metabolic analysis showed that the external morphology, protein expression profile, and ginsenoside synthesis ability of the F. Ginseng increased just like that of W. Ginseng with the increase in age. Our study represents the first characterization of the proteome of F. Ginseng during development and provides new insights into the metabolism and accumulation of ginsenosides.