Screening method for cDNAs encoding putative enzymes converting loganin into secologanin by a transgenic yeast culture

A screening method was developed for the detection of enzymes converting loganin to secologanin, a precursor in the biosynthesis of indole alkaloids. The method uses a transgenic yeast culture expressing two cDNAs encoding enzymes involved in the terpenoid indole alkaloid biosynthesis. In the presence of secologanin, the yeast culture produces a yellow compound visible on nitrocellulose. This color change was used to screen a cDNA library of Catharanthus roseus for a putative enzyme converting loganin into secologanin.     

Effects of alkaloid precursor feeding and elicitation on the accumulation of secologanin in a Catharanthus roseus cell suspension culture

In a Catharanthus roseus cell line accumulating secologanin, time-course studies on the uptake of loganin and the in vivo conversion to secologanin were performed. Four-day-old cells converted 100% of the fed loganin to secologanin within 24 hours, showing that this step is unlikely to be limiting for alkaloid accumulation. Thirteen-day-old cells also took up loganin, but only about 25% was recovered as secologanin. A saturation in the uptake of loganin and in the conversion of loganin into secologanin was observed after feeding increasing amounts of loganin. Elicitation by cellulase and pectinase decreased the cellular contents of secologanin and strictosidine whereas it increased the tryptamine content. In addition, the uptake of loganin in elicited cells was blocked. In vitro assays with protein extracts of elicited Catharanthus roseus cells indicated the activation of secologanin degrading enzyme(s). Feeding of tryptophan did not result in any increase in alkaloid contents, despite its complete uptake. Tryptamine feeding led to increased strictosidine contents, but ajmalicine levels remained unchanged. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.     

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.     

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells. Received: 25 February 1996 / Revision received: 16 August 1996 / Accepted: 30 September 1996     

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.     

Purification and properties of strictosidine synthetase (an enzyme condensing tryptamine and secologanin) from Catharanthus roseus cultured cells.

Strictosidine synthetase, which catalyzes the condensation of tryptamine with secologanin to form strictosidine (isovincoside), was purified 740-fold to homogeneity from cultured cells of Catharanthus roseus in 10% yield. The specific activity is 5.85 nkat/mg. The molecular weight as estimated by gel filtration is 38,000. The isoelectric point is 4.6. Apparent Km values for tryptamine and secologanin are 0.83 and 0.46 mM, respectively. The enzyme shows a broad pH optimum between 5.0 and 7.5. The product of the enzymic reaction is exclusively strictosidine, while no trace of its epimer vincoside can be detected. Sulfhydryl inhibitors have no effect on the enzyme. End products in the biosynthetic pathway of indole alkaloids such as ajmalicine, vindoline, and catharanthine do not inhibit the activity of strictosidine synthetase.     

Quantification of artemisinin in Artemisia annua extracts by 1H-NMR

Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent. (1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the using the delta 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2) = 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI.     

Radioimmunoassay for the determination of loganin and the biotransformation of loganin to secologanin by plant cell cultures

A radioimmunoassay technique has been developed for the quantitative measurement of loganin in crude extracts from both fresh and dried material of whole plants and cultivated plant cells. The assay makes use of 6′-carboxyloganin which is rendered immunogenic through linkage to bovine serum albumin. The tracer molecule was synthesized via periodate opening of the glucose moiety of loganin and subsequent reduction with sodium borotritide of high specific activity. The rabbit antibodies had a high affinity (K_a = 1.6 × 10⁹1/mol) for loganin and permitted the detection of as little as 0.1 ng per 0.05 ml of sample. The antiserum was highly specific for loganin and its aglycone, with only 10-hydroxyloganin and 7-epi-loganin showing a substantial cross reactivity. A number of cell cultures of the Caprifoliaceae were tested for their ability to transform added loganin to secologanin. By labelled precursor feeding experiments members of the genera Weigelia, Lonicera, Hydrangea and Symphoricarpus were found to open the cyclopentane ring of loganin. The time course of the biotransformation of loganin was monitored using radioimmunoassays for both loganin and secologanin and cell cultures of Lonicera tatarica as biological material.     

Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.     

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.     

Quantitative analysis of ginkgolic acids from Ginkgo leaves and products using 1H-NMR

The determination of ginkgolic acids in Ginkgo products is one of the principal components of quality control. However, a number of ginkgolic acids with different side chains may be present and this makes their analysis by conventional chromatographic methods more complex. In this study, 1H-NMR spectrometry was applied to the analysis of the total content of ginkgolic acids in leaves of Ginkgo biloba and in six types of commercial Ginkgo products in the absence of chromatographic purification. For this analysis, protons H-3, H-4, and H-5, which are well separated in the range 8 (ppm) 6.5-7.5 in the 1H-NMR spectrum, were utilised. For further confirmation, the correlations of H-3, H-4 and H-5 were examined by 1H-1H COSY spectra in all extracts. The quantity of the compounds was calculated from the relative ratio of the integral of each peak to the integral of the peaks of a known amount (100 microg) of anthracene used as an internal standard. The quantitative results obtained by 1H-NMR analysis were compared with those obtained by GC, which showed that the 1H-NMR method allows a simple quantification of total ginkgolic acids in Ginkgo extracts without any pre-purification steps.     

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat’s pellets

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.     

Measurement of propericiazine in plasma by capillary gas chromatography and selected ion monitoring detection

A new method, based upon a selective extraction and gas chromatographic/mass spectrometric analysis, was developed to monitor the effect of combined haemoperfusion-haemodialysis treatment in a case of propericiazine poisoning. The method relies on the selected ion monitoring of the acetate derivatives of propericiazine and its internal standard fluphenazine, after their extraction from 1 ml of alkalinized plasma with n-hexane:isopropanol (8:2, v/v), back-extraction into an acidified water phase, realkalinization and extraction with n-hexane: isopropanol, derivatization with acetic anhydride and gas chromatography on a short (12 m) OV-101 fused silica capillary column. The described procedure is specific and provides between-assay variability of 4.8% CV at 5 micrograms 1(-1) plasma concentration. The method enables quantification down to 1 microgram 1(-1) and hence demonstrates sufficient sensitivity to permit pharmacokinetic or drug monitoring studies.     

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8?cells?μL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.     

Optimisation of supercritical fluid extraction of indole alkaloids from Catharanthus roseus using experimental design methodology--comparison with other extraction techniques

Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation, was applied to optimise supercritical fluid extraction (SFE) conditions for the extraction of indole alkaloids from the dried leaves of Catharanthus roseus. The effects of pressure (200-400 bar), temperature (40-80 degrees C), modifier concentration (2.2-6.6 vol%) and dynamic extraction time (20-60 min) on the yield of alkaloids were evaluated. The extracts were analysed by high-performance liquid chromatography and the analytes were identified using ion trap-electrospray ionisation-mass spectrometry. The method was linear for alkaloid concentration in the range 0.18-31 microg/mL. The limits of detection and quantification for catharanthine, vindoline, vinblastine and vincristine were 0.2, 0.15, 0.1 and 0.08 microg/mL and 2.7, 2.0, 1.3 and 1.1 microg/g, respectively. The dry weight content of major alkaloids in the plants were compared using different extraction methods, i.e. SFE, Soxhlet extraction, solid-liquid extraction with sonication and hot water extraction at various temperatures. The extraction techniques were also compared in terms of reproducibility, selectivity and analyte recoveries. Relative standard deviations for the major alkaloids varied from 4.1 to 17.5% in different extraction methods. The best recoveries (100%) for catharanthine were obtained by SFE at 250 bar and 80 degrees C using 6.6 vol% methanol as modifier for 40 min, for vindoline by Soxhlet extraction using dichloromethane in a reflux for 16 h, and for 3',4'-anhydrovinblastine by solid-liquid extraction using a solution of 0.5 m sulphuric acid and methanol (3:1 v/v) in an ultrasonic bath for 3 h.     

Optimisation of a microwave-assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Introduction – Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. Objectives – The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. Matrial and methods – The WA from the air‐dried aerial part of Withania somnifera Dunal. was extracted using a microwave‐assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high‐performance thin‐layer chromatography assay was validated and applied for the quantification of each experiment. Results – The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2‐propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter‐day, and intra‐day RSDs of the developed method indicated the validity of the method. Conclusion – A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial‐based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio. Copyright © 2010 John Wiley & Sons, Ltd.     

液质联用多反应监测法定量目标多肽或蛋白质

为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 （MRM）分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析.     

A rapid method for analysis of abscisic acid (ABA) in crude extracts of water stressed Arabidopsis thaliana plants by liquid chromatography--mass spectrometry in tandem mode.

We describe a quick, simple method for the extraction and quantification of the phytohormone (+)-abscisic acid (ABA) in samples of plants subjected to different water deficit treatments. The method includes an extraction with acetone/water/acetic acid (80:19:1, v/v), evaporation of the extracts and finally injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) system in multiple reaction monitoring (MRM) mode. The objective of this work has been to show the applicability of the method to quantify the endogenous content of ABA in Arabidopsis thaliana (Columbia, Col-0) leaves at three different degrees of water stress. Control plants, had almost constant low levels of ABA (2-3 ng g-1, f.w.) throughout the 3 weeks of the experiment. Nevertheless, stressed plants increase the ABA content between the first and the second week (from 10 to 21 ng g-1, f.w.). The results suggest that this method is useful for quantifying ABA from plant material and that it avoids tedious and time-consuming extraction, purification and/or derivatization processes.