Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

Colorimetric recognition of the coralyne-poly(dA) interaction using unmodified gold nanoparticle probes, and further detection of coralyne based upon this recognition system

Among the functional nucleic acids studied, adenine-rich nucleic acids have attracted attention due to their critical roles in many biological processes and self-assembly-based nanomaterials, especially deoxyribonucleic acids (abbreviated as poly(dA)). Therefore the ligands binding to poly(dA) might serve as potential therapeutic agents. Coralyne, a kind of planar alkaloid, has been firstly found that it could bind strongly to poly(dA). This work herein reports an approach for visual sensing of the coralyne-poly(dA) interaction. This method was based on the coralyne inducing poly(dA) into the homo-adenine DNA duplex and the difference in electrostatic affinity between single-stranded DNA and double-stranded DNA with gold nanoparticles (GNPs). Furthermore, we applied the recognition process of the interaction between coralyne and poly(dA) into specific coralyne detection with the assistance of certain software (such as Photoshop). A linear response from 0 to 728 nM was obtained for coralyne, and a detection limit of 91 nM was achieved.

利用电喷雾质谱技术研究复方中药的配伍原则

Traditional Chinese Medicine (TCM) based on natural products is one important part of the Chinese civilization.Owing to the complexity of the composition,the study on medical effective components and curative effects are very difficult;Fuzi (Radix Aconiti Lateralis Praeparata) has been widely used for cardiotonic and analgesics in China,however,the component-aconitine in which is very toxic and may cause some side-effect.This paper reported the application of electrospray iohization mass spectrometric(ESIMS) technique on study of the compound prescriptions containing fuzi,Renshensini concoction and Baweidihuang concoction,to explore the mechanism of synergy between fuzi and other herbs.From chemical point of view,the detoxification mechanism can be attributed to the hydrolysis of the solubility of toxic diester-alkaloids from concoctions of fuzi.Radix Glycyrrhizae Praeparata,Rhizoma Zingiberis,Radix Ginsengp promote the hydrolysis to produce the less toxic monoester-alkaloids and Fructus Corni reduce the solubility of hypaconitine from fuzi.

Structural elucidation and identification of alkaloids in Rhizoma Coptidis by electrospray ionization tandem mass spectrometry

Simple, convenient, sensitive and accurate analytical methods are needed for the structural characterization and identification of alkaloid components in Rhizoma Coptidis in traditional Chinese herbal medicine, which has important bioactivity. In this work, the identification of alkaloid compounds in Rhizoma Coptidis was investigated by obtaining molecular mass information using electrospray ionization mass spectrometry (ESI-MS). Multi-stage tandem mass spectrometric (ESI-MSn) data for the alkaloid compounds were used for detailed structural characterization, then structure information was obtained by comparison of the fragmentation mechanisms of both alkaloids in Rhizoma Coptidis and standard samples of berberine, palmatine, coptisine and jatrorrhizine by MS. Based on the results obtained, the structure of a novel compound was elucidated. The results of the experiments demonstrate that ESI-MSn is a sensitive, selective and effective tool for the rapid determination of alkaloids in Rhizoma Coptidis.

The use of CE-electrochemiluminescence with ionic liquid for the determination of bioactive constituents in Chinese traditional medicine

CE/tris(2,2-bipyridyl) ruthenium(ll) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL), CEECL, with an ionic liquid (IL) detection system was established for the determination of bioactive constituents in Chinese traditional medicine opium poppy which contain large amounts of coexistent substances. A minimal sample pretreatment which involves a one-step extraction approach avoids both sample loss and environmental pollution. As the nearby hydroxyl groups in some alkaloid such as morphine may react with borate to form complexes and IL, as a high-conductivity additive in running buffer, could cause an enhanced field-amplified effect of electrokinetic injection. Running buffer containing 25 mM borax-8 mM 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) IL (pH 9.18) was used which resulted in significant changes in separation selectivity and obvious enhancement in ECL intensities for those alkaloids with similar structures. Sensitive detection could be achieved when the distance between the Pt working electrode and the outlet of separation capillary was set at 150 mu m and the stainless steel cannula was fixed approximately 1 cm away from the outlet of the capillary. Quantitative analysis of four alkaloids was achieved at a detection voltage of 1.2 V and a separation voltage of 15 kV in less than 7 min.

Analysis of strychnos alkaloids using electrospray ionization Fourier transform ion cyclotron resonance multi-stage tandem mass spectrometry

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.

Comparison of two marine sponge-associated Penicillium strains DQ25 and SC10: differences in secondary metabolites and their bioactivities

Two strains of Penicillium, DQ25 and SC10, isolated from marine sponge Haliclona angulata (Bowerbank) and Hymeniacidon sp. respectively, were subjected to stationary cultivation under GYP medium for 30 days. The fermentation extracts were undergone bioactivities assays against human pathogens, phytopathogenic fungi and brine shrimp (Artemia salina). Bioassays-guided compounds isolation was performed by Silica gel columns and Sephadex LH-20 chromatography. Spectroscopic methods were used to structures elucidation of the compounds. Results showed the activities of secondary metabolites of strain DQ25 were generally stronger than that of strain SC10. Major bioactive molecules isolated from strain DQ25 were a 1,4-naphthoquinone derivative and an unidentified alkaloid. The two components were not isolated from the extract of strain SC10. ITS sequences revealed that these two species have the greatest similarity with Penicillium vinaceum and Penicillium granulatum respectively.

Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve

Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.

Two new bromoindoles from red alga Laurencia similis

Two new bromoindole alkaloids have been isolated from the ethanolic extract of the red alga Laurencia similis. On the basis of chemical and spectroscopic methods (including 1D and 2D NMR technique), their structures have been elucidated as 2,2',5,5',6,6'-sixibromo-3, 3'-bi-1H-indole and 3, 5-dibromo-1-methylindole, respectively. (C) 2008 Xiao Bin Zhu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Comparative Study of Contents of Four Tropane Alkaloids in Cultural and Wild Anisodus tanguticus

To assess the medicinal value of cultural Anisodus tanguticus,the contents of four bioactive tropane alkaloids,anisodine,anisodamine,scopolamine and atropine,in cultural and wild materials were determined by the HPLC method.The results showed that content of each alkaloid in the aboveground parts of cultural and wild samples was lower than that in roots,and this explained why it was not the whole plant but the root that was used as medicinal materials.The content of each alkaloid in the roots of one-year cultural material was lower than that in the two-year plants.The discrepancy of the total of four alkaloids between one-year and wild plants is not significant.Moreover,the total of four alkaloids,and the contents of anisodine,scopolamine,and atropine in two-year plants were higher than those in wild plant.Thus there is medicinal value in the cultivated A.tanguticus as well as wild A.tanguticus,especially in the two-year cultural A.tanguticus.

Study on the interaction between strychnine and bovine serum albumin by capillary electrophoretic frontal analysis

The protein binding constant, binding sites of the Strychnos alkaloid-strychnine and bovine serum albumin (BSA) was determined by capillary electrophoretic frontal analysis (CE-FA) for the first time. The experiment was carried out in a polyacrylamide-coated fused silica capillary (48.4 cmx50 mu m i.d., 38.1 cm effective length) with 20 mmol/L citrate/MES buffer (pH 6.0, ionic strength 0.17). The applied voltage was 12 kV and detection wavelength was set at 257 nm. The plateau height of the peak was employed to determine the unbound concentration of drug in BSA equilibrated sample solution based on the external drug standard in the absence of protein. The present method provides a convenient, accurate technique for the early stage of drug screening.