Ajmalicine metabolism in Catharanthus roseus cell cultures (original) (raw)
Applied Microbiology and Biotechnology, 1993
In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine sYnthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the lowdensity culture had accumulated six times more ajmalicine (in gmoles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between highand low-density cultures were in the AS and TDC activities, which were two to three times higher in the lowdensity culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.
Biotechnology Progress, 2003
The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 µM) and a low concentration of BA (2.22 µM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 µM) and a high concentration of BA (8.90 µM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 µM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 µM) supplemented medium compared with the high concentration of BA (8.90 µM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 µM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.
Biotechnology and Bioengineering, 2002
The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e ∼70% and also resulted in the complete recovery of ajmalicine from the cell. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 408–415, 2002.
Influence of Fungal Elicitors on Production of Ajmalicine by Cell Cultures of Catharanthus roseus
Biotechnology Progress, 2002
Suspension cultures of Catharanthus roseus ( C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A.niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T.viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A.niger, F. moniliforme, and T. viride. The maximum ajmalicine production (75 μg g−1 dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 μg g−1 DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 μg g−1 DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 μg g−1 DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.
Scaleup of ajmalicine production by plant cell cultures ofCatharanthus roseus
Biotechnology and Bioengineering, 1993
The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of kLa, aeration rate, CO2 production rate, and influent gas phase CO2 concentration on the liquid phase CO2 concentration are discussed. © 1993 John Wiley & Sons, Inc.
Applied Biochemistry and Biotechnology, 1987
The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungi Chrysosporium pahnorum, Eurotium rubrum, Micromucor isabellina, and Pythium aphanidermatum when aseptically added to cell suspensions of Cantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 ~g/L ajmalicine and 600 p,g/L catharanthine were detected in C. roseus cell suspension grown in the presence of the M. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.
Biochemical Engineering Journal, 2004
The potential of strategies for increasing secondary metabolite accumulation from plant cell cultures may still be partially limited by unfavorable conditions for biosynthesis (i.e. feedback inhibition, unfavorable equilibrium state) or product metabolism. In this paper, the rate of product removal was altered and used as a tool for probing the impact of these limiting conditions on product accumulation, using the production of ajmalicine and serpentine from Catharanthus roseus suspensions as a model system. The method for altering the in situ product removal rate was simple; Amberlite ® XAD-7HP resin was enclosed in either a porous or less porous material, and experiments confirmed that the product removal rate was higher with resin enclosed in the porous Miracloth versus the less porous nylon.
Effect of culture process on alkaloid production by Catharanthus roseus cells
Journal of Biotechnology, 1991
The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced ~ 20 g dw per I of biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (> 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg 1-~ and 25 to 4000 /xg per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in produc-Correspondence to: J. Archambault, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Canada H4P 2R2. Abbreviations: 2,4 D: 2,4 dichlorophenoxyacetic acid; APM: alkaloid production medium; AB5:B5 growth medium with IAA; B5: plant cell basal growth medium; KLa: mass transfer coefficient; TIA: total indole alkaloid content; SM: secondary metabolites. 2 tion. Small but significant (~ 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.
Effect of a tertiary amine on alkaloid accumulation in Catharanthus roseus
Phytochemistry, 1997
The Madagascar periwinkle, Catharanthus roseus, produces numerous indole alkaloids, several of which have important therapeutic activities. These secondary metabolites are among the most expensive drugs because of their low abundance in intact plants. We investigated the biotransformation of a synthetic product (N-(methoxycarbonylethyl)-N-[2-(1H-indol-3yl)-ethyl]-fl-methyl alaninate) in the roots and aerial parts of C. roseus cultivated in vitro, in order to stimulate de novo the biosynthesis of biologically active indole alkaloids. The results showed a large increase (90%) of ajmalicine in the roots whilst serpentine and yohimbine levels were unchanged. In the aerial parts, the synthetic product was recovered and the time courses of various alkaloids were unchanged. No new molecule was observed. The four alkaloids tested increased with plant age whilst yohimbine gradually decreased.
Journal of biotechnology, 1991
The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced ~ 20 g dw per I of biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (> 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg 1-~ and 25 to 4000 /xg per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in produc
Plant Cell Reports, 1991
The addition of Aspergillus niger homogenate to Catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. The use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. Furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. The exposure of the cells to an osmotic stress produced a marked increment (320%) in the total alkaloid content. When both stress treatments were applied sequentially, an additive effect on alkaloid accumulation was observed. It was 300% higher than in cells cultured without treatment, the majority of the alkaloids found in the medium.
Content of Ajmalicine on Cultured Callus Catharanthus roseus (L.) G. Don with Tryptophan Treatment
2017
Catharanthus roseus (L.) G. Don is cultivated as an ornamental plant and also has a high economic value because it contains terpenoid indole alkaloids such as ajmalicine that have pharmacological benefits. This research was conducted to investigate the content of ajmalicine in callus culture that was given tryptophan treatment with concentrations of 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L and 250 mg/L. Identification and analysis of ajmalicine using HPLC Shimpak VP-ODS C18 150 x 4.6 mm at a wavelength of 254 nm. The results showed that the ajmalicine content was lower after the tryptophan treatment, where the minimum ajmalicine content of the tryptophan 200 mg/L, ie 0.346 μg/g dw. Ajmalicine content of the treatment of 250 mg / L tryptophan, is the highest content in all treatments, ie 2.686 μg/g bk. So it can be concluded that the treatment of tryptophan in callus culture Catharanthus roseus can decrease the content of ajmalicine.
Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies
Journal of Biotechnology, 1991
Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid 'release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 /zg l-l), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HC1 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (< 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increases in total alkaloid (from ~ 5-10 mg 1-1 to 15 mg l-l), ajmalicine (from 0 to ~ 0.29 mg 1-1) Correspondence to: J. Archambault, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Canada H4P 2R2. Abbreviations: B5: plant cell basal growth medium; APM: alkaloid production medium; AB5:B5 medium containing IAA; IAA: indole acetic acid; IA: immobilized airlift bioreactor; KLa: mass transfer coefficient (h-l); TIA: total indole alkaloids.
Plant Science, 2006
The effects of triadimefon, a triazole compound on the antioxidant potentials and root alkaloid ajmalicine content were studied in two varieties, rosea and alba of Catharanthus roseus (L.) G. Don., an important medicinal plant. The plants of both the varieties were subjected to 15 mg l−1 triadimefon treatment by soil drenching on 53, 68 and 83 days after planting (DAP). The plants were harvested on 60, 75 and 90 days after planting and the antioxidant potentials and ajmalicine content were estimated. The antioxidant potentials viz., ascorbic acid (AA), α-tocopherol and reduced glutathione (GSH) were found increased under triadimefon treatment. The antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities showed slight changes in both the varieties under triadimefon treatment when compared to control plants. Indole alkaloid ajmalicine content increased significantly under triadimefon treatment. The increase in ajmalicine content was more in rosea variety than in alba variety. These preliminary results suggest that, the application of triadimefon may be a useful tool to increase the alkaloid production in medicinal plants.
2017
Periwinkle (Catharanthus roseus (L.) G. Don) is a medicinal plant containing about 130 types of alkaloids that have important pharmacological effects. Ajmalicine in periwinkle root is an antihypertensive drug used in treatment of high blood pressure. Adventitious roots obtained from periwinkle leaves of in vitro shoots grew well in quarter-strength MS medium supplemented with 0.3 mg/l IBA and 20 g/l sucrose. Dark condition was more suitable for root growth than light. However, callus formation also took place in addition to the growth of adventitious roots. Temporary immersion system was applied in the culture of adventitious roots in order to reduce the callus growth rate formed in shake flask cultures. The highest growth index of roots was achieved using the system with 5-min immersion every 45 min (1.676 ± 0.041). The roots cultured in this system grew well without callus formation. Ajmalicine content was highest in the roots cultured with 5-min immersion every 180 min (950 μg/g dry weight).
Pharmaceutical Biology, 2016
Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli.
Journal of Biotechnology, 1994
Bioreactor systems have been developed for the production of ajmalicine, an alkaloid used in the treatment of hypertension. Cell cultures of Catharanthus roseus produced higher levels of ajmalicine (323/zg g-1 dry weight) in a production medium enriched with tryptophan. The cell cultures were grown in medium prepared in tap water and market sugar with a view to minimise the costs of production. Large-scale cultivation of cell suspension was performed in a 20-1 airlift bioreactor under controlled conditions. An ajmalicine production of 315/~g g-~ dry weight was achieved in the bioreactor after 14 d of cultivation.
Revista Brasileira De Ciencia Do Solo, 2018
Catharanthus roseus (L) G. Don (Madagascar periwinkle) belongs to the Apocynaceace family and is widely spread throughout tropical and subtropical regions of the world. The plant produces several important alkaloids, such as ajmalicine and serpentine, which are used in the treatment of circulatory diseases. The potential of inoculation with arbuscular mycorrhizal fungi (AMF) and nitrogen fertilization to enhance the production of alkaloids was investigated in periwinkle. A greenhouse experiment was carried out to evaluate the effects of arbuscular mycorrhizal fungi and N fertilizer dosages on plant growth, production of ajmalicine, and nutrient content in roots. The concentration of ajmalicine was determined by reverse-phase high-performance liquid chromatography with UV detection. The experiment was designed in randomized blocks in a 4 × 4 factorial scheme with four microbiological treatments (control -without mycorrhiza; Claroideoglomus etunicatum; Rhizophagus intraradices; mixed inoculum -Rhizophagus clarus + Gigaspora margarita), and four N fertilizer dosages (15, 30, 60, and 120 mg kg -1 ) with four replications. Catharanthus roseus growth was higher when plants were inoculated with mixed inoculum (R. intraradices + G. margarita) and C. etunicatum. The mixed inoculum (R. intraradices + G. margarita) and C. etunicatum, combined with N fertilization, enhanced ajmalicine yield. Catharanthus roseus inoculated with mycorrhiza showed increased P absorption and reduced N content.
Callus and biomass culture of Catharanthus roseus L. were established to check for the presence of total alkaloid and its subsequent yield. Various treatments like strength of nutrient salts, sucrose concentrations and combinations of plant growth regulators (PGR's) were applied to both MS and B5 in agar as well as suspension medium to test their effects on enhanced alkaloid content and its yield. There was no significant difference in any of the observable parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield if the culture were treated similarly in both types of media formulations (MS or B5 salts). Physical state (agar solidified or the liquid suspension) of the medium had significant effect on all the parameters particular on fresh wt, alkaloid content and yields. Although, the fresh wt. and dry wt. of biomass in suspension culture was 2-3 times less than that of callus obtained from agar medium. However, the alkaloid content and yield was 2-3 times higher in suspension culture compared to agar medium in similar treatments. The highest alkaloid content observed was 5.67mg/g dwt in B5 suspension medium containing 3% sucrose and modified with 0.5mg/l 2,4-Dichlorophenoxy acetic acid (2,4-D) + 1 mg/l Kinetin (KIN) + 2mg/l α-naphthalene acetic acid (NAA). The combined effects of these factors on the enhanced production of total alkaloids were expected to contain higher yield of anticancer vinblastine and vincristine in the cell suspension culture system.