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.

A new spermidine macrocyclic alkaloid isolated from Gymnosporia arenicola leaf.

The isolation and structural elucidation of a macrocyclic alkaloid, characterized by the presence of a 13-membered macrolactam ring containing a spermidine unit N-linked to a benzoyl group is hereby reported. The structure of this previously unknown spermidine alkaloid isolated from Gymnosporia arenicola (Celastraceae) leaves has been elucidated by (1)H and (13)C NMR spectroscopy (including bidimensional analysis) and further characterized by high-resolution mass spectrometry and polarimetry. A route for the biosynthesis of this new bioactive macrocycle is proposed and the cytotoxicity of the compound was evaluated against two ATCC cell lines - one normal-derived (MCF10A) and one cancer-derived cell line (MCF7) - using the MTT assay. The alkaloid revealed to be non-cytotoxic against both cell lines. The IC50 values from the cells were also determined.

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.

Isolation and identification of spermidine derivatives in tea (Camellia sinensis) flowers and their distribution in floral organs.

BACKGROUND: Recently, tea (Camellia sinensis) flowers have attracted increasing interest because of their content of bioactive compounds such as catechins. The aim of this study was to investigate the occurrence of some characteristic compounds in tea flowers. RESULTS: A principal component analysis of metabolites using ultra-performance liquid chromatography/time-of-flight mass spectrometry showed differences in metabolite profile between flowers and leaves of C. sinensis var. Yabukita. Four spermidine derivatives were isolated from tea flowers. One of them was determined as N(1) ,N(5) ,N(10) -tricoumaroyl spermidine based on NMR, MS and UV data. The other three were identified as feruoyl dicoumaroyl spermidine, coumaroyl diferuoyl spermidine and triferuoyl spermidine based on MS(n) data. Tricoumaroyl spermidine as the major spermidine conjugate was not detected in tea leaves. Furthermore, it decreased during floral development and mainly occurred in anthers. CONCLUSION: This study has provided the first evidence that spermidine-phenolic acid conjugates occur in tea flowers in considerable amounts. Their presence should prompt a reconsideration of the ecological role of tea flowers. From an economic point of view, tea flowers might be suitable as a raw material in the healthcare food and pharmaceutical industries.

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.

Dracotanosides A-D, spermidine glycosides from Dracocephalum tanguticum: structure and amide rotational barrier.

Four new spermidine glycosides, dracotanosides A-D (1-4), have been isolated from Dracocephalum tanguticum. These molecules represent the first spermidine glycosides from this plant genus. The structures, including absolute configurations, were determined by spectroscopic and chemical methods. The amide bond rotational barrier of aglycone 1a was calculated by density functional theory (DFT) computation.

New spermidines from the florets of Carthamus tinctorius.

Two new spermidine compounds, namely safflospermidine A (1) and safflospermidine B (2), together with two known compounds, N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (3) and N(1),N(5),N(10)-(E)-tri-p-coumaroylspermidine (4), were isolated from the florets of Carthamus tinctorius L. Their structures were elucidated by spectroscopic means.

N1,N5,N10-Tris(4-hydroxycinnamoyl)spermidines from Microdesmis keayana roots.

Three new N1,N5,N10-tris(4-hydroxycinnamoyl)spermidines were isolated from a methanolic root extract of Microdesmis keayana. They were identified as N5,N10-di(p-coumaroyl)-N1-feruloylspermidine,N5-(p-coumaroyl)-N1,N10-diferuloylspermidine, and N1,N5,N10-triferuloylspermidine, and were named keayanidines A, B, and C (1-3), respectively. Their structures were established by spectral techniques(electrospray mass spectrometry, one- and two-dimensional NMR). A 4',4'',4'''-trimethylated derivative was prepared by methylation of keayanidine C, and the same compound was synthesized fromspermidine and 3,4-dimethoxycinnamic acid to confirm the spectral attributions of the NMR data of the natural compounds. Radical-scavenging properties of all compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical spectrophotometric assay.

Antifungal activity of three spermidine conjugates.

Three tri-substituted spermidines, di-p-coumaroyl-caffeoylspermidine, tri-caffeoylspermidine and tri-p-coumaroylspermidine, isolated from pollen of Quercus alba, were examined for antifungal activity. Both di-p-coumaroyl-caffeoylspermidine and tri-p-coumaroylspermidine reduced mycelial growth of the oat leaf stripe pathogen, Pyrenophora avenae and reduced powdery mildew (Blumeria graminis f. sp. hordei) infection of barley seedlings when applied as a post-inoculation treatment. When used as a pre-inoculation treatment, only di-p-coumaroyl-caffeoylspermidine reduced powdery mildew infection significantly. Growth of P. avenae in the presence of 100 microM di-p-coumaroyl-caffeoylspermidine reduced activity of S-adenosylmethionine decarboxylase (AdoMetDC), and led to a reduction in the incorporation of labelled ornithine into spermidine. The other two spermidine conjugates increased AdoMetDC activity and the flux label from ornithine into spermine in P. avenae significantly.

Fast-growing root nodule bacteria produce a novel polyamine, aminobutylhomospermidine.

Polyamines in various root nodule bacteria including Bradyrhizobium japonicum, Rhizobium fredii, R. leguminosarum, R. meliloti and R. loti were identified by capillary gas chromatography. Homospermidine was the polyamine present in highest concentration in all the rhizobia tested. In addition to putrescine and homospermidine, fast-growing type of rhizobial cells contained a novel polyamine, aminobutylhomospermidine, NH2(CH2)4NH(CH2)4NH(CH2)4NH2. The unusual tetraamine was not found in the cells of slow-growing type of rhizobia throughout their growth period, indicating a difference in polyamine metabolism between fast-growing type and slow-growing type of root nodule bacteria.