Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hydroxyethyl jasmonate

Chemically synthesized 2-hydroxyethyl jasmonate (HEJA) was for the first time employed to induce the ginsenoside biosynthesis and to manipulate the product heterogeneity in plant cell cultures. The dose response and timing of HEJA elicitation were investigated in cell suspension cultures of Panax notoginseng. The optimal concentration and timing of HEJA addition for both cell growth and ginsenoside accumulation was identified to be 200 μM added on day 4. It was interestingly found that HEJA could stimulate ginsenosides biosynthesis and change their heterogeneity more efficiently than methyl jasmonate (MJA), i.e., the total ginsenoside content and the Rb/Rg ratio increased about 60 and 30% with HEJA elicitation than that by MJA, respectively. The activity of Rb₁ biosynthetic enzyme, i.e., UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), was also higher in the former case. A maximal production titer of ginsenoside Rg₁, Re, Rb₁, and Rd was 47.4±4.8, 52.3±4.4, 190±18, and 12.1±2.5 mg/l with HEJA elicitation, which was about 1.3-, 1.3-, 1.7-, and 2.1-fold than that using MJA, respectively. Early signal events in plant defense response, including oxidative burst and jasmonic acid (JA) biosynthesis, were also examined. Levels of H₂O₂ and NO in medium and l-phenylalanine ammonia lyase activity in cells were not affected by addition of MJA and HEJA. On the other hand, the JA content in cells was increased with external jasmonates elicitation, and it was inhibited with the addition of JA biosynthesis inhibitors. The results suggest that oxidative burst might not be involved in the jasmonates-elicited signal transduction pathway, and MJA and HEJA may induce the ginsenoside biosynthesis via induction of endogenous JA biosynthesis and key enzymes (such as UGRdGT) in the ginsenoside biosynthetic pathway of P. notoginseng cells. The information is useful for hyperproduction of plant-specific heterogeneous products.     

Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation

A single addition of 200 M methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume). Repeated elicitation with two additions of 200 M MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels. The content of ginsenosides Rg₁, Re, Rb₁ and Rd in the ALR was increased from, respectively, 0.18±0.01, 0.21±0.01, 0.21±0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32±0.02, 0.36±0.02, 0.72±0.06 and 0.08±0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43±0.02, 0.46±0.03, 1.09±0.07 and 0.14±0.02 mg per100 mg DW with two additions of MJA. Interestingly, the activity of the Rb₁ biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb₁ content. In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted. The final cell density and total ginsenoside content in the ALR reached 27.3±1.5 g/l and 2.02±0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg₁, Re, Rb₁ and Rd was 111.8±4.7, 117.2±4.6, 290.2±5.1 and 32.7±8.1 mg/l, respectively. The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.     

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     

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.     

A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of l-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 M PFPJA on day 7 led to a high Tc content (38.2±0.3 mg/g) at day 21, while the Tc content was 29.3±0.3 mg/g and 34.9±0.9 mg/g with the addition of 100 M MJA and TFEJA, respectively. Quantitative structure–activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.     

Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.     

Novel synthetic jasmonates as highly efficient elicitors for taxoid production by suspension cultures of Taxus chinensis

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.     

Impact of methyl jasmonate on squalene biosynthesis in microalga Schizochytrium mangrovei

Squalene is an effective antioxidant and a potential chemopreventive agent. In this work, the effect of methyl jasmonate (MJA) on squalene biosynthesis in microalga Schizochytrium mangrovei was investigated. The maximum squalene content (1.17 ± 0.06 mg/g cell dry weight, DW) reached during the next 3 h after MJA treatment (0.1 mM) at 48 h of cultivation, which was 60% higher than that of control. The activity of squalene synthase (SS) increased 2-fold over control at this point. The maximum cholesterol content of 0.45 ± 0.03 mg/g DW was reached at hour 51 when MJA concentration was 0.4 mM, whereas the squalene content was lower at this point. The observations suggested that the increased squalene content was resulted from an increased activity of SS. MJA could be used to regulate the key enzymes in squalene biosynthetic pathway for the increased production of this compound in thraustochytrids. This research also provided novel information on the stimulation effect of methyl jasmonate on the biosynthesis of essential intermediate involved in the primary metabolism in microorganism.     

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 M); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 M MJ. In the second experiment, 200 M MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.     

Endophytes isolated from Panax notoginseng converted ginsenosides

Endophytes may participate in the conversion of metabolites within medicinal plants, influencing the efficacy of host. However, the distribution of endophytes within medicinal plants P. notoginseng and how it contributes to the conversion of saponins are not well understood. Here, we determined the distribution of saponins and endophytes within P. notoginseng compartments and further confirm the saponin conversion by endophytes. We found metabolites showed compartment specificity within P. notoginseng. Potential saponin biomarkers, such as Rb1, Rg1, Re, Rc and Rd, were obtained. Endophytic diversity, composition and co-occurrence networks also showed compartment specificity, and bacterial alpha diversity values were highest in root compartment, consistently decreased in the stem and leaf compartments, whereas those of fungi showed the opposite trend. Potential bacterial biomarkers, such as Rhizobium, Bacillus, Pseudomonas, Enterobacter, Klebsiella, Pantoea and fungal biomarkers Phoma, Epicoccum, Xylariales, were also obtained. Endophytes related to saponin contents were found by Spearman correlation analysis, and further verification experiments showed that Enterobacter chengduensis could convert ginsenoside Rg1 to F1 at a rate of 13.24%; Trichoderma koningii could convert ginsenoside Rb1 to Rd at a rate of 40.00% and to Rg3 at a rate of 32.31%; Penicillium chermesinum could convert ginsenoside Rb1 to Rd at a rate of 74.24%.     

Application of ultra-performance LC-TOF MS metabolite profiling techniques to the analysis of medicinal <Emphasis Type="Italic">Panax</Emphasis> herbs

The morphological appearance and some ingredients of Panax ginseng, Panax notoginseng and Panax japonicus of the Panax genus are similar. However, their pharmacological activities are obviously different due to the significant differences in the types and quantity of saponins in each herb. In the present study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) was used to profile the abundances of metabolites in the three medicinal Panax herbs. Multivariate statistical analysis technique, that is, principle component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discriminate between the Panax samples. PCA of the analytical data showed a clear separation of compositions among the three medicinal herbs. The critical markers such as chikusetsusaponin IVa, ginsenoside R0, ginsenoside Rc, ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rg2 accountable for such variations were identified through the corresponding loading weights, and the tentative identification of biomarkers is completed by the accurate mass of TOFMS and high resolution and high retention time reproducibility performed by UPLC. The proposed analytical method coupled with multivariate statistical analysis is reliable to analyze a group of metabolites present in the herbal extracts and other natural products. This method can be further utilized to evaluate chemical components obtained from different plants and/or the plants of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150 microM and cultured for 40 days. Up to 100 microM MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 microM MJ peaked after 10 days at 48 mg g(-1) dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.     

Biotransformation of Saponins by Endophytes Isolated from Panax notoginseng

The biotransformation of the major saponins in Panax notoginseng, including the ginsenosides Rg1, Rh1, Rb1, and Re, by endophytes isolated from P. notoginseng was studied. One hundred and thirty‐six endophytes were isolated and screened for their biotransformational abilities. The results showed that five of the tested endophytes were able to transform these saponins. These five strains were identified based on their ITS or 16S rDNA sequences, which revealed that they belonged to the genera Fusarium, Nodulisporium, Brevundimonas, and Bacillus genera. Ten transformed products were isolated and identified, including a new compound 6‐O‐[α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]‐20‐O‐β‐D ‐glucopyranosyldammarane‐3,6,12,20,24,25‐hexaol ( 3 ), and nine known compounds, compound K ( 1 ), ginsenoside F2 ( 2 ), vinaginsenoside R13 ( 4 ), vinaginsenoside R22 ( 5 ), pseudo‐ginsenoside RT4 ( 6 ), (20S)‐protopanaxatriol ( 7 ), ginsenoside Rg1 ( 8 ), vinaginsenoside R15 ( 9 ), and (20S)‐3‐O‐β‐D ‐glucopyranosyl‐6‐O‐β‐D ‐glucopyranosylprotopanaxatriol ( 10 ). This is the first study on the biotransformation of chemical components in P. notoginseng by endophytes isolated from the same plant.     

Inhibitory Effects of Jasmonic Acid and its Analogues on Barley (Hordeum vulgare L.) Anther Extrusion

Plant hormones play many important roles in plant growth and development. We previously tried to control barley (Hordeum vulgare L.) flowering in ear cultures treated with plant hormones and related compounds, and found that anther extrusion from florets was reduced by treatment with 100 ppm methyl jasmonate (MJA). In the present study, we show that 10 ppm MJA also inhibits anther extrusion in barley ear cultures. Spraying field-grown barley ears with MJA clarified the efficacy of this chemical under field conditions, but higher concentrations (100 to 1000 ppm MJA) were required. In addition, we investigated the activity of jasmonic acid (JA) analogues and cucurbic acid analogues in barley ear cultures. Some esters of JA showed similar effects to that of MJA, but effects of 9,10-dihydrojasmonic acid (DJA) and cucurbic acid derivatives were lower than that of MJA. In light of these data, we discuss the structural requirements for increased inhibition of barley anther extrusion.     

Improvement of cephalomanine production in Taxus chinensis cells by a combination of sucrose and methyl jasmonate

Cell suspension cultures of Taxus chinensis, supplemented with 25 g sucrose l^–1, produced 11 mg cephalomanine l^–1, 21 g biomass l^–1 and 19 nkat geranylgeranyl diphosphate (GGPP) synthase activity g protein^–1. Supplementation of the cultures with 100 M methyl jasmonate (MJA) produced 17 mg cephalomanine l^–1, 6 g biomass l^–1 and 78 nkat GGPP synthase activity g protein^–1. Addition of sucrose and MJA together produced 24 mg cephalomanine l^–1, 18 g biomass l^–1 and 55 nkat GGPP synthase activity g protein^–1.     

Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng

Adventitious roots ofPanax ginseng C.A. Meyer (a natural tetraploid) were treated with 50 or 100 mg L^-1 colchicine for 12, 24,36, 48, or 60 h to induce polyploid (octoploid) roots. The largest number of octoploid roots was obtained with a 100 mg L^-1 colchicine treatment over 60 h. To verify that ginsenoside was being accumulated in the developing tissues, the tetraploid (control) and octoploid roots were cultured for 40 d in Murashige and Skoog media that lacked NH₄NO₃ but was supplemented with 2 mg L^-1 naphthaleneacetic acid and 50 g L_-1 sucrose. Levels of fresh and dry biomass were greater in the octoploid roots. Although total ginsenoside and Rb-group ginsenoside contents were less in the octoploid roots than in the tetraploids, the former had a higher amount of Rg-group ginsenosides (especially Rg1). These results demonstrate the benefit that polyploid adventitious roots provide in enhancing the production of secondary metabolites in ginseng.