Biotransformation of ferulic acid and vanillylamine to capsaicin and vanillin in immobilized cell cultures of Capsicum frutescens

Plant tissue culture medium which contained FeEDTA as sole iron source was incubated aseptically in light (16-h photoperiod, 100 mol m^-2 s^-1 PAR) at 20°C without plant tissue. Soluble iron dropped from an initial concentration of 4 mg 1^-1 to less than 0.1 mg 1^-1 in 4 weeks. This occurred in both glass and plastic culture vessels. No loss occurred when medium was incubated at 20°C in darkness. A further experiment showed that soluble iron concentration fell to <0.2 mg 1^-1 in only 4 days but the loss was slower at lower irradiances.Effects of the loss of soluble iron on plantlet growth were assessed by culturing single node stem segments of in vitro potato (Solanum tuberosum L. cv. Arran Banner) plantlets on medium previously exposed to light. Pre-exposure sufficient to reduce soluble iron concentration to <0.1 mg 1^-1 had no inhibitory effect on plantlet development in solidified medium or in liquid medium, except when the liquid medium had been centrifuged before inoculation to remove iron precipitated during pre-exposure to light. The plantlets then became chlorotic.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

The accumulation of phenylpropanoid and capsaicinoid compounds in cell cultures and whole fruit of the chilli pepper,Capsicum frutescens Mill

The accumulation of the phenylpropanoid precursors of capsaicin in suspended and immobilised cell cultures of C. frutescens has been studied and compared with accumulation in whole pepper fruit. The use of HPLC techniques has revealed that the phenolic precursors of capsaicin are present in chilli pepper cells at extremely low levels, irrespective of the source of tissue or the developmental state. Radioactive tracer studies have indicated that the majority of the phenolic derivatives of phenylalanine are ultimately bound to the insoluble fraction of the cells. Results from experiments where immobilised cell cultures were grown under conditions which enhance capsaicin yield would suggest that the diversion of compounds into this bound fraction has a considerable influence upon capsaicin biosynthesis in this system.     

Multivariate optimisation of microwave-assisted extraction of capsaicin from Capsicum frutescens L. and quantitative analysis by 1H-NMR

A simple and rapid microwave-assisted extraction (MAE) procedure combined with 1H-NMR spectrometry was developed and optimised for the extraction and quantitative determination of capsaicin in Capsicum frutescens. The influence of experimental variables, including irradiation power, extraction temperature and dynamic extraction time before reaching the selected extraction temperature, on the performance of the extraction procedure was systematically studied using a Box-Behnken experimental design followed by a conventional central composite design approach. Statistical treatment of the results together with results from some additional experiments suggested optimum extraction conditions as 120 degrees C and 150 W, for 15 min with acetone as extractant. The optimised MAE method provides extracts that can be analysed quantitatively using 1H-NMR without any preliminary clean-up or derivatisation steps. In the 1H-NMR spectrum of the crude extracts the doublet signal in the delta range 4.349-4.360 ppm was well separated from other resonances in deuterated chloroform. The quantity of the compound was calculated from the relative ratio of the integral value of the target peak to that of a known amount of dimethylformamide as internal standard. In comparison with traditional Soxhlet extraction, the proposed method is less labour-intensive and provides a drastic reduction of extraction time and solvent consumption. In addition, MAE showed higher extraction yield and selectivity, with comparable reproducibility and recovery, relative to both conventional Soxhlet and sonication methods.     

Biotransformation of protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in freely suspended and immobilized cell cultures of Capsicum frutescens

Freely suspended cells and immobilized cell cultures of Capsicum frutescens Mill. were treated with phenylpropanoid intermediates--protocatechuic aldehyde and caffeic acid to study their biotransformation ability. It was found that externally fed protocatechuic aldehyde and caffeic acids were biotransformed to vanillin and capsaicin. It was noted that this culture biotransformed externally fed protocatechuic aldehyde to vanillin more than its conversion to capsaicin, whereas, caffeic acid-treated cultures accumulated more capsaicin than vanillin. The maximum accumulation of vanillin (5.63 mg l(-1)) and capsaicin (3.83 mg l(-1)) was recorded on the 6th and 15th day, respectively in immobilized C. frutescens cell cultures treated with protocatechuic aldehyde, which was 1.8 and 1.4 times higher than in protocatechuic aldehyde-treated freely suspended cell cultures. Caffeic acid-treated immobilized C. frutescens cell cultures accumulated maximum vanillin and capsaicin at 2.68 and 3.03 mg l(-1) culture, respectively, on the 9th and 12th day, which was 1.65 and 1.33 times over freely suspended cultures treated with caffeic acid. The addition of S-adenosyl-L-methionine, a methyl donor, to protocatechuic aldehyde-treated immobilized C. frutescens cell cultures, resulted in accumulation of vanillin (14.08 mg l(-1)) on the 4th day, which was 2.5-fold higher than that in cultures treated with protocatechuic aldehyde alone, suggesting the influence of S-adenosyl-L-methionine on O-methylation of protocatechuic aldehyde, resulting in more vanillin accumulation. The increase in vanillin accumulation was well correlated with an increase in specific activity of caffeic acid O-methyltransferase in protocatechuic aldehyde and S-adenosyl-L-methionine-treated immobilized C. frutescens cell cultures. This study also provides an example for an alternative route to formation of vanillin by C. frutescens cell cultures.     

Regulatory effects of exogenous branched-chain amino acids in Nicotiana plumbaginifolia cell suspension cultures

Nicotiana plumbaginifolia suspension cultured cells were grown on medium supplemented with valine, leucine and isoleucine, singly or in combination. The effects of the three branched-chain amino acids on cell growth rate and on the activity of acetohydroxyacid synthase (AHAS), the first enzyme (and the main regulative site) of their biosynthetic pathway, were studied. Results showed that valine and leucine, at concentrations ranging from 10^–4 to 10^–3 M, inhibit growth, and at higher doses (from 10^–2 to 10^–1 M) AHAS activity. Growth, but not AHAS activity, was affected also by isoleucine. The addition of ammonium succinate to the culture medium, in order to counteract a possible general inhibitory effect of these compounds on nitrogen metabolism, relieved only partially their cytotoxicity. Feeding cells with equimolar mixtures of the three amino acids resulted in a minor but reproducible decrease in AHAS level, which was proportional to the dose. A similar result was obtained also on N. plumbaginifolia seedlings, suggesting that in this species a modulation of enzyme level could play a role in controlling the flow of metabolites through the pathway.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acids - FAD flavin adenine dinucleotide - GS glutamine synthetase - TPP thiamine pyrophosphate     

An unexpected effect of capsaicin on spontaneous GABA-ergic IPSCS in hippocampal cell cultures

Vanilloid receptors 1 (VRs1) expressed in a subpopulation of sensory neurons and responsible for processing of chemical and thermal noxious stimuli were also shown to be expressed in several cerebral structures and to be involved in the regulation of glutamatergic synaptic transmission. In this study, we started to investigate the possibility that VRs1 are also involved in the regulation of GABA-ergic synaptic transmission. For this purpose, the effect of a VR1 agonist, capsaicin, on spontaneous GABA-ergic inhibitory postsynaptic currents (IPSCs) was studied in hippocampal cell cultures using a patch-clamp technique. It was found that capsaicin (10 μM) decreased both the frequency and amplitude of spontaneous IPSCs. This finding suggests the involvement of VRs1 in the regulation of neuronal firing in some GABA-ergic interneurons and in the modulation of the efficacy of GABA-ergic synaptic transmission. However, considering the direction of the effect (a decrease in the IPSC frequency) and lack of its desensitization, the involvement of other receptor(s) also cannot currently be ruled out. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 364–367, July–August, 2006.     

The influence of intracellular pools of phenylalanine derivatives upon the synthesis of capsaicin by immobilized cell cultures of the chilli pepper,Capsicum frutescens

In an attempt to determine the potential factors controlling the biosynthesis of the secondary metabolite capsaicin by immobilized cell cultures of the chilli pepper, Capsicum frutescens Mill, labelling techniques using the radioactive precursor [¹⁴C]phenylalanine have been employed. Following preincubation treatments with either capsaicin (the end-product of the pathway) or sinapic acid, [¹⁴C]phenylalanine was applied and the movement of the label through the pathway and its eventual fate was followed. Results have shown that capsaicin, through a feedback-inhibition mechanism, negatively influences its own synthesis. Furthermore, capsaicin synthesis in these cells is not controlled via the activity of the enzymes phenylalanine ammonia-lyase and cinnamate 4-hydroxylase which may determine the rate of entry of metabolites into the phenylpropanoid pathway. The importance of other sinks for phenylalanine derivatives, which may compete for capsaicin precursors, has also been investigated. Surprisingly, protein proved to be only a relatively minor sink for phenylalanine with the great majority of the label rapidly ending up in covalently bound phenolics in the cell wall. Attempts to prevent this by applying sinapic acid were only partially successful. The importance of these results in relation to the possible control mechanisms which operate to control secondary metabolite synthesis in vitro is discussed.     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Putrescine carbamoyltransferase activity in pea is associated with the ornithine carbamoyltransferase polypeptide and is not involved in putrescine synthesis under physiological conditions

Putrescine carbamoyltransferase (PutCT) has been postulated to function in the synthesis of putrescine (Put) from an N-carbamoylputrescine (NCPut) intermediate in plants. In pea, PutCT activity was associated entirely with ornithine carbamoyltransferase (OCT) protein, which was purified to homogeneity using an immobilized transition-state analog inhibitor ( N-(phosphonacetyl)-L-ornithine). No evidence for a separate PutCT enzyme, similar to that in Streptococcus [15], or PutCT activity associated with a putrescine synthase-type multifunctional enzyme [13] was found. OCT carried out the carbamoylation of Put and other diamine and polyamine substrates inefficiently and at non-physiological pH (Put carbamoylation: pH 10.8 optimum, V_max 0.11 kat/mg protein, K_m=6.7 mM for Put and 1.0 mM for carbamoyl-P), when compared with ornithine carbamoylation (pH 8.5 optimum, V_max=313.9 kat/mg protein, K_m=4.4 mM for ornithine and 0.6 mM for carbamoyl-P). Different subcellular compartmentation of PutCT activity (chloroplast) and the NCPut substrate (cytosol), coupled with a thermodynamically-unfavorable reverse reaction (i.e., Put synthesis from NCPut), suggest that the OCT-associated PutCT activity does not significantly contribute to in vivo Put synthesis in plants.Abbreviations ADC Arginine Decarboxylase - Agm Agmatine - AIH Agmatine Iminohydrolase - Cad Cadaverine - Cit Citrulline - CP Carbamoyl-P - Dap 1,3-Diaminopropane - Dns- Dansyl- - NCPut N-Carbamoylputrescine - OCT Ornithine Carbamoyltransferase - Put Putrescine - PutCT Putrescine Carbamoyltransferase - Spd Spermidine - Spm Spermine     

葡萄细胞悬浮培养生产白藜芦醇

以巨峰葡萄果皮为外植体,在添加2.0 mg/L 6-苄基嘌呤（6-BA）和0.1 mg/L 2,4-二氯苯氧基（2,4-D）的B5培养基上诱导葡萄愈伤组织; 以50 g/L的初始接种量在添加1.0 mg/L 6-BA和0.05 mg/L 2,4-D的B5液体培养基上建立葡萄悬浮培养体系。在25～27 ℃下,摇床振荡暗培养（120～130 r/min）18 d后,葡萄细胞生物量和白藜芦醇含量达到最大值（16.17 g/L、95.69 μg/g干质量）。在培养第12天时,向培养基中添加100 μmol/L茉莉酸甲酯（MeJA）,经过6 d处理,细胞中白藜芦醇含量达235.73 μg/g干质量。     

Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures ofCapsicum frutescens and activity of phenylalanine ammonia lyase

Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.     

Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili

Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g^?1 f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g^?1 f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g^?1 f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g^?1 f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g^?1 f.wt on day 20 and 1,315.3 ± 10 μg g^?1 f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.     

Regulation of sucrose storage by amino acid arginine in sugarcane cell suspension cultures

Sugarcane cell cultures were obtained from callus formed on explants derived from young expanding leaves of two early maturing sugarcane varieties viz “CoJ83” and “CoJ86”. The cell cultures were varied with different arginine concentrations in the culture medium. For each cultivar, sucrose content with 20 μM arginine in the culture medium decreased from 3 to 5 days and then increased to 10 days after subculturing. Higher concentration of arginine in the culture medium (60 μM) decreased the sucrose content at different days after subculturing and thus significantly stimulated sucrose mobilization. The activity of sucrose synthase and sucrose phosphate synthase reached maximum while the activity of acid and neutral invertase was minimal in the culture medium with 20 μM arginine. Thus arginine at low concentration (20 μM) enables the cells to accumulate the higher level of sucrose. The optimum level of amino acids can be utilized to regulate the in vivo activity of sucrose synthase, sucrose phosphate synthase and invertase to achieve maximum sucrose accumulation in sugarcane storage tissue.     

Improved plant regeneration in maize callus cultures after pretreatment with dl-alpha-difluoromethylarginine

The influence of a three-month-long pretreatment with dl-alpha-difluoromethylarginine (DFMA), an irreversible suicide inhibitor of arginine decarboxylase activity (ADC; EC 4.1.1.19), on plant regeneration, protein and polyamine (PA) composition of Zea mays callus cultures has been investigated. A four-fold increase in the number of regenerated plants is obtained after pretreatment with 0.5 mM DFMA. In addition, the regeneration frequency increases 3-fold in the treated calluses and the plants regenerated from such cultures are more developed than the untreated controls. The data obtained on protein and PA contents suggest that a senescence effect is exerted on the calluses grown in the presence of DFMA. However, after DFMA removal a rejuvenation effect occurs on the calluses that may explain the improvement of morphogenic capacity. This study indicates that DFMA pretreatment can be used to increase regeneration efficiency from maize callus cultures.     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua

Artemisinin, isolated from the shrub-Artemisia annua, is a sesquiterpene lactone used to treat multi-drug resistant strains of falciparum malaria. It is also effective against a wide variety of cancers such as leukemia and colon cancer. To counter the present low content in leaves and uneconomical chemical synthesis, alternate ways to produce artemisinin have been sought. But this compound remains elusive in cell cultures of A. annua despite the extensive studies undertaken. This work reports the first successful approach for production of artemisinin by cell cultures of Indian variety of A. annua. In the present study, an integrated yield enhancement strategy, developed by addition of selected precursor (mevalonic acid lactone) and elicitor (methyl jasmonate) at optimized concentrations, resulted in 15.2g/l biomass and 110.2mg/l artemisinin, which was 5.93 times higher in productivity in comparison to control cultures.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.