Separation and Characterization of Phenolamines and Flavonoids from Rape Bee Pollen, and Comparison of Their Antioxidant Activities and Protective Effects Against Oxidative Stress.

Phenolamines and flavonoids are two important components in bee pollen. There are many reports on the bioactivity of flavonoids in bee pollen, but few on phenolamines. This study aims to separate and characterize the flavonoids and phenolamines from rape bee pollen, and compare their antioxidant activities and protective effects against oxidative stress. The rape bee pollen was separated to obtain 35% and 50% fractions, which were characterized by HPLC-ESI-QTOF-MS/MS. The results showed that the compounds in 35% fraction were quercetin and kaempferol glycosides, while the compounds in 50% fraction were phenolamines, including di-p-coumaroyl spermidine, p-coumaroyl caffeoyl hydroxyferuloyl spermine, di-p-coumaroyl hydroxyferuloyl spermine, and tri-p-coumaroyl spermidine. The antioxidant activities of phenolamines and flavonoids were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays. It was found that the antioxidant activity of phenolamines was significantly higher than that of flavonoids. Moreover, phenolamines showed better protective effects than flavonoids on HepG2 cells injured by AAPH. Furthermore, phenolamines could significantly reduce the reactive oxygen species (ROS), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and increase the superoxide dismutase (SOD) and glutathione (GSH) levels. This study lays a foundation for the further understanding of phenolamines in rape bee pollen.

Ecological Insights to Track Cytotoxic Compounds among Maytenus ilicifolia Living Individuals and Clones of an Ex Situ Collection.

Biodiversity is key for maintenance of life and source of richness. Nevertheless, concepts such as phenotype expression are also pivotal to understand how chemical diversity varies in a living organism. Sesquiterpene pyridine alkaloids (SPAs) and quinonemethide triterpenes (QMTs) accumulate in root bark of Celastraceae plants. However, despite their known bioactive traits, there is still a lack of evidence regarding their ecological functions. Our present contribution combines analytical tools to study clones and individuals of Maytenus ilicifolia (Celastraceae) kept alive in an ex situ collection and determine whether or not these two major biosynthetic pathways could be switched on simultaneously. The relative concentration of the QMTs maytenin (1) and pristimerin (2), and the SPA aquifoliunin E1 (3) were tracked in raw extracts by HPLC-DAD and ¹H-NMR. Hierarchical Clustering Analysis (HCA) was used to group individuals according their ability to accumulate these metabolites. Semi-quantitative analysis showed an extensive occurrence of QMT in most individuals, whereas SPA was only detected in minor abundance in five samples. Contrary to QMTs, SPAs did not accumulate extensively, contradicting the hypothesis of two different biosynthetic pathways operating simultaneously. Moreover, the production of QMT varied significantly among samples of the same ex situ collection, suggesting that the terpene contents in root bark extracts were not dependent on abiotic effects. HCA results showed that QMT occurrence was high regardless of the plant age. This data disproves the hypothesis that QMT biosynthesis was age-dependent. Furthermore, clustering analysis did not group clones nor same-age samples together, which might reinforce the hypothesis over gene regulation of the biosynthesis pathways. Indeed, plants from the ex situ collection produced bioactive compounds in a singular manner, which postulates that rhizosphere environment could offer ecological triggers for phenotypical plasticity.

Polyamines interfere with protein ubiquitylation and cause depletion of intracellular amino acids: a possible mechanism for cell growth inhibition.

Spermidine is a polyamine present in eukaryotes with essential functions in protein synthesis. At high concentrations spermidine and norspermidine inhibit growth by unknown mechanisms. Transcriptomic analysis of the effect of norspermidine on the plant Arabidopsis thaliana indicates upregulation of the response to heat stress and denatured proteins. Accordingly, these polyamines inhibit protein ubiquitylation, both in vivo (in yeast, Arabidopsis, and human Hela cells) and in vitro (with recombinant ubiquitin ligase). This interferes with protein degradation by the proteasome, a situation known to deplete cells of amino acids. Norspermidine treatment of yeast cells induces amino acid depletion, and supplementation of media with amino acids counteracts growth inhibition and cellular amino acid depletion but not inhibition of protein polyubiquitylation.

Enhancement of Glucose Utilization by Loesenerine through AMPK Activation in Myotubes.

Type 2 diabetes is characterized by hyperglycemia derived from insulin resistance in periphery tissue. Effects of skeletal muscle on glucose disposal are closely related to insulin resistance. The potential effects on mitochondrial function of loesenerine, a macrocyclic spermidine alkaloid from the aerial part of Euonymus fortunei (TURCZ.) HAND.-MAZZ were observed after a high-throughout screening based on mitochondrial membrane potential (MMP) assay. Further pharmacological studies revealed that loesenerine activates AMP-activated protein kinase (AMPK) pathway through increasing ADP/ATP ratio by inhibiting mitochondrial respiration. In addition, loesenerine induced 1.07-, 1.14-, and 1.22-fold increment of glucose uptake in C2C12 cells at the concentrations of 20, 40 and 80 µmol/L, respectively. Meanwhile, incubated with loesenerine for 12 h increased glucose consumption in a dose-dependent manner in C2C12 cells. This is the first report that macrocyclic spermidine alkaloid possesses potential hypoglycemic activity in vitro.

Polyamines are required for normal growth in Sinorhizobium meliloti.

Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

Identification of potential trypanothione reductase inhibitors among commercially available ß-carboline derivatives using chemical space, lead-like and drug-like filters, pharmacophore models and molecular docking.

American trypanosomiasis or Chagas disease caused by the protozoan Trypanosoma cruzi (T. cruzi) is an important endemic trypanosomiasis in Central and South America. This disease was considered to be a priority in the global plan to combat neglected tropical diseases, 2008-2015, which indicates that there is an urgent need to develop more effective drugs. The development of new chemotherapeutic agents against Chagas disease can be related to an important biochemical feature of T. cruzi: its redox defense system. This system is based on trypanothione ([Formula: see text],[Formula: see text]-bis(glutathyonil)spermidine) and trypanothione reductase (TR), which are rather unique to trypanosomes and completely absent in mammalian cells. In this regard, tricyclic compounds have been studied extensively due to their ability to inhibit the T. cruzi TR. However, synthetic derivatives of natural products, such as [Formula: see text]-carboline derivatives ([Formula: see text]-CDs), as potential TR inhibitors, has received little attention. This study presents an analysis of the structural and physicochemical properties of commercially available [Formula: see text]-CDs in relation to compounds tested against T. cruzi in previously reported enzymatic assays and shows that [Formula: see text]-CDs cover chemical space that has not been considered for the design of TR inhibitors. Moreover, this study presents a ligand-based approach to discover potential TR inhibitors among commercially available [Formula: see text]-CDs, which could lead to the generation of promising [Formula: see text]-CD candidates.

Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 µM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 µM.

Epilithonimonas ginsengisoli sp. nov., isolated from soil of a ginseng field.

A novel Gram-staining-negative, rod-shaped bacterium, designated DCY78(T), was isolated from soil of a ginseng field in Yeon-cheon province (38° 04' 00″ N 126° 57' 00″ E), Republic of Korea. The phylogenetic analysis based on 16S rRNA gene sequences showed that strain DCY78(T) belonged to the genus Epilithonimonas and was most closely related to Epilithonimonas lactis DSM 19921(T) (98.5 % sequence similarity) and Epilithonimonas tenax DSM 16811(T) (97.8 %). Growth occurred at 10-30 °C with an optimum temperature of 28 °C. The pH range for growth was pH 5.5-8.0. The major polar lipids were found to be phosphatidylethanolamine three unidentified amino lipids and one unidentified polar lipid. The only predominant quinone was MK-6. The major polyamines were sym-homospermidine and spermidine. The major fatty acids were summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c), iso-C15 : 0 and iso-C17 : 0 3-OH. The DNA G+C content was 37.9 mol%. On the basis of the phenotypic and genotypic analysis, the isolate is classified as representative of a novel species in the genus Epilithonimonas, for which the name Epilithonimonas ginsengisoli is proposed. The type strain is DCY78(T) ( = KCTC 32174(T) = JCM 19896(T)).

Hephaestia caeni gen. nov., sp. nov., a novel member of the family Sphingomonadaceae isolated from activated sludge.

A Gram-staining-negative, rod-shaped and motile bacterium, designated strain ERB1-3(T), was isolated from a laboratory-scale activated sludge system treating coke plant effluent using thiocyanate-supplemented growth medium. Strain ERB1-3(T) was oxidase-positive and weakly catalase-positive. The predominant fatty acids were C18:1ω7c (35.6 %) and C17:1ω6c (29.2%), and the major respiratory quinone was Q-10. Polar lipids were dominated by sphingoglycolipid and phosphatidylglycerol. Major polyamines were spermidine and sym-homospermidine. The G+C content of the genomic DNA of strain ERB1-3(T) was 66.4 mol%. Based on the 16S rRNA gene, strain ERB1-3(T) exhibited the highest sequence similarity values to Sphingomonas sanxanigenens DSM 19645(T) (96.1%), Sphingobium scionense DSM 19371(T) (95.1%) and Stakelama pacifica LMG 24686(T) (94.8%) within the family Sphingomonadaceae. The novel isolate had some unique chemotaxonomic features that differentiated it from these closely related strains, contained much more C17 : 1ω6c, C15 : 0 2-OH, C17:0 and C17:1ω8c fatty acids and possessed diphosphatidylglycerol only in trace amounts. On the basis of the phenotypic, chemotaxonomic and molecular data, strain ERB1-3(T) is considered to represent a novel genus and species, for which the name Hephaestia caeni gen. nov., sp. nov. is proposed. The type strain is ERB1-3(T) ( = DSM 25527(T) = NCAIM B 02511(T)).

Chryseobacterium camelliae sp. nov., isolated from green tea.

A Gram-staining-negative, strictly aerobic, non-motile, rod-shaped and flexirubin-type-pigmented strain, THG C4-1(T), was isolated from green tea leaves in Jangheung-gun, Republic of Korea. Strain THG C4-1(T) grew well at 20-30 °C, at pH 7.0-7.5 and in the absence of NaCl on nutrient agar. Based on 16S rRNA gene sequence comparisons, strain THG C4-1(T) was most closely related to Chryseobacterium taiwanense Soil-3-27(T) (97.7 %), C. hagamense RHA2-9(T) (97.2 %), C. gregarium P 461/12(T) (97.2 %), C. ginsenosidimutans THG 15(T) (97.1 %), C. taeanense PHA3-4(T) (97.0 %) and C. daeguense K105(T) (97.0 %), but DNA-DNA relatedness between strain THG C4-1(T) and its closest phylogenetic neighbours was below 21 %. The DNA G+C content was 41.7 mol%. The only isoprenoid quinone detected in strain THG C4-1(T) was menaquinone 6 (MK-6). The major component of the polyamine pattern was sym-homospermidine. The major polar lipids were phosphatidylethanolamine and unidentified aminolipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. These data supported the affiliation of strain THG C4-1(T) to the genus Chryseobacterium. The results of physiological and biochemical tests enabled strain THG C4-1(T) to be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. Therefore, the novel isolate represents a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed, with THG C4-1(T) ( = KACC 16985(T) = JCM 18745(T)) as the type strain.

Mygalin: a new anticonvulsant polyamine in acute seizure model and neuroethological schedule.

Polyamines are compounds that interact with ionotropic receptors, mainly modulating the NMDA receptor, which is strictly related to many neurologic diseases such as epilepsy. Consequently, polyamines rise as potential neuropharmacological tools in the prospection of new therapeutic drugs. In this paper, we report on the biological activity of synthetic polyamine Mygalin, which was tested as an anticonvulsant in model of chemically induced seizures. Male Wistar rats were injected with vehicle, diazepam, MK-801 or Mygalin at different doses followed by Pentylenetetrazole or N-Methyl-D-Aspartate administration. Mygalin presented protection against seizures induced by both NMDA injections and PTZ administration by 83.3% and 16.6%, respectively. Moreover, it prolonged the onset of tonic-clonic seizures induced by PTZ. Furthermore, it was tested in neuroethological schedule evaluating possible side-effects and it presented mild changes in Open Field, Rotarod and Morris Water Maze tests when compared to available anticonvulsant drugs. The mechanism underlying the anticonvulsant effect of Mygalin is noteworthy of further investigation, nevertheless, based on these findings, we hypothesize that it may be wholly or in part due to a possible NMDA receptor antagonism. Altogether, the results demonstrate that Mygalin has an anticonvulsant activity that may be an important tool in the study of prospection of therapeutics in epilepsy neuropharmacology.

Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR.

Sphingomonas kyungheensis sp. nov., a bacterium with ginsenoside-converting activity isolated from soil of a ginseng field.

A bacterial strain THG-B283(T), which has ß-glucosidase activity, was isolated from soil of a ginseng field. Cells were Gram-reaction-negative, oxidase- and catalase-positive, aerobic, motile with one polar flagellum and rod-shaped. The strain was subjected to a polyphasic taxonomic study. Strain THG-B283(T) grew optimally at around pH 7.0, at 25-28 °C and in the absence of NaCl on R2A agar. 16S rRNA gene sequence analysis revealed that strain THG-B283(T) belongs to the family Sphingomonadaceae and is closely related to Sphingomonas melonis DAPP-PG 224(T) (98.2 %), S. aquatilis JSS7(T) (98.1 %), S. insulae DS-28(T) (97.6 %), S. mali IFO 15500(T) (97.1 %) and S. pruni IFO 15498(T) (97.0 %). Strain THG-B283(T) contained Q-10 as the predominant ubiquinone. The major fatty acids included summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω7c, C14 : 0 2-OH and C16 : 0. The DNA G+C content was 72.2 mol%. The major component in the polyamine pattern was sym-homospermidine. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, sphingoglycolipid, diphosphatidylglycerol, an unidentified glycolipid, unidentified aminolipids, an unidentified phospholipid and unidentified lipids. Genomic and chemotaxonomic data supported the affiliation of strain THG-B283(T) to the genus Sphingomonas. DNA-DNA relatedness between strain THG-B283(T) and its closest phylogenetic neighbours was below 23 %. On the basis of phenotypic, phylogenetic and genetic data, strain THG-B283(T) represents a novel species of genus Sphingomonas, for which the name Sphingomonas kyungheensis sp. nov. is proposed. The type strain is THG-B283(T) ( = KACC 16224(T) = LMG 26582(T)).

Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N5,N¹°-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N5,N¹°-di-(Z,E)-feruloylspermidine and N¹-acetyl-N5,N¹°-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N5,N¹°-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

Hymenobacter ginsengisoli sp. nov., isolated from soil of a ginseng field.

A Gram-stain-negative, non-motile, red bacterium, designated DCY57(T), was isolated from soil of a ginseng field in a mountainous region of Chungnam province in South Korea. Strain DCY57(T) grew with 0-1 % (w/v) NaCl and the optimum temperature for growth was 30 °C. Strain DCY57(T) contained MK-7 as the predominant menaquinone. The polyamine was sym-homospermidine. The major fatty acids were C(16:1)ω5c, iso-C(15:0), anteiso-C(15:0) and summed feature 3 (containing C(16:1)ω7c and/or C(16:1)ω6c). The major polar lipids were phosphatidylethanolamine, unknown aminophospholipids, unknown aminolipids and unknown lipids. The DNA G+C content was 58.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DCY57(T) was most closely related to members of the genus Hymenobacter. The isolate exhibited 91.7 % 16S rRNA gene sequence similarity with H. soli PB17(T), 94.5 % with H. flocculans A2-50A(T) and 95.8 % with H. metalli A2-91(T). On the basis of the evidence presented in this study, strain DCY57(T) represents a novel species within the genus Hymenobacter, for which the name Hymenobacter ginsengisoli sp. nov. is proposed. The type strain is DCY57(T) ( = KCTC 23674(T) = JCM 17841(T)).

Hippocampal polyamine levels and transglutaminase activity are paralleling spatial memory retrieval in the C57BL/6J mouse.

Although a potential role for polyamines and transglutaminases (TGs) in memory mechanisms have been proposed, hippocampal spermine (SPM) and spermidine (SPD) levels as well as transamidating activity of TG in spatial memory have not been addressed yet. It was therefore the aim of the study to assess hippocampal polyamines and TG activity at the probe trial in a spatial memory paradigm. C57BL/6J mice (20 animals per group) were used for the experiments and divided into a trained and a yoked (untrained) group. The Morris water maze (MWM) was selected as the memory test, animals were sacrificed within 5 min following the probe trial and hippocampi were taken for biochemical analysis. SPD and SPM levels were assessed by an analytical procedure according to Gismondi et al. Transamidating activity of TG was determined following the method described by Chung and Folk using [14C] methylamine as substrate. γ-(Glutamyl)-polyamine levels were evaluated by ion exchange chromatography according to Folk et al. Animals learned the task in the MWM as latencies and pathlengths were significantly reduced. At the probe trial mice showed significantly higher preference for the target quadrant. Free SPD and SPM levels were manifold decreased in the trained as compared to the yoked group. Transamidating activity of TG was fourfold increased in trained as compared to yoked controls. γ-(Glutamyl)-SPD was comparable while γ-(glutamyl)-SPM was significantly higher in the trained group. The findings show a potential role for polyamines, their derivative γ-(glutamyl)-SPM and transamidating activity of TG at memory retrieval or formation. Results from this study are extending and knowledge on polyamines and report for the first time involvement of γ-(glutamyl)-SPM and transamidating activity of TG that may form the basis for future neurochemical and pharmacological studies and indeed, modulation of polyamine and TG activity has been already proposed as a tentative therapeutical concept.

Deciphering molecular mechanism underlying antileishmanial activity of Nyctanthes arbortristis, an Indian medicinal plant.

ETHNOPHARMACOLOGICAL RELEVANCE: Nyctanthes arbortristis L. (Oleaceae) is widely used in the traditional medicine of India. The plant is shown to have antibacterial and antileishmanial activities. AIM OF THE STUDY: Evaluation of iridoid glucosides from the plant as inhibitor of trypanothione reductase (TryR), a validated drug target enzyme of the Leishmania parasite. The study contributes towards understanding mechanism of antileishmanial effect of the plant. MATERIALS AND METHODS: TryR of Leishmania parasite is expressed and purified. Iridoid glucosides are isolated from the plant and tested as inhibitor of TryR enzyme of the parasite. RESULTS: Inhibitory constant (K(i)) of various iridoid glucosides ranges from 3.24±0.05 µM to 6.49±0.05 µM. Thus, the molecular mechanism underlying antileishmanial activity of these compounds is mediated through inhibition of TryR. CONCLUSION: The current study also points out towards potential application of iridoid glucosides as novel drugs against the disease.

Evolution of a novel phenolic pathway for pollen development.

Metabolic plasticity, which largely relies on the creation of new genes, is an essential feature of plant adaptation and speciation and has led to the evolution of large gene families. A typical example is provided by the diversification of the cytochrome P450 enzymes in plants. We describe here a retroposition, neofunctionalization, and duplication sequence that, via selective and local amino acid replacement, led to the evolution of a novel phenolic pathway in Brassicaceae. This pathway involves a cascade of six successive hydroxylations by two partially redundant cytochromes P450, leading to the formation of N1,N5-di(hydroxyferuloyl)-N10-sinapoylspermidine, a major pollen constituent and so-far-overlooked player in phenylpropanoid metabolism. This example shows how positive Darwinian selection can favor structured clusters of nonsynonymous substitutions that are needed for the transition of enzymes to new functions.

A novel compound N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition.

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC(50) of 0.74+/-0.15 microM for S6 cells or 1.07+/-0.23 microM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.