Jan Schripsema

Application of NMR in Plant Metabolomics: Techniques, Problems and Prospects †

The present state-of-the-art of NMR in plant metabolomics is reviewed. Attention is paid to the different practical aspects of the application of NMR. The sample preparation, the measurement of the spectrum, quantitative aspects and data analysis are discussed. Each stage has its specific problems, which are pointed out and recommendations are made.

Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

Antibacterial and antifungal properties of wax and hexane extracts of Citrus spp. peels were tested using bioautographic and microdilution techniques against three plant pathogenic fungi (Penicillium digitatum, Curvularia sp., and Colletotrichum sp.), two human pathogens (Trichophyton mentagrophytes and Microsporum canis), and two opportunistic bacteria (Escherichia coli and Staphylococcus aureus). Two polymethoxylated flavonoids and a coumarin derivative, were isolated and identified from peel extracts, which presented antimicrobial activity especially against M. canis and T. mentagrophytes: 4,5,6,7,8-pentamethoxyflavone (tangeritin) and 3,4,5,6,7,8-hexamethoxyflavone (nobiletin) from C. reticulata; and 6,7-dimethoxycoumarin (also known as escoparone, scoparone or scoparin) from C. limon.

Acaricidal properties of the essential oil and precocene II obtained from Calea serrata (Asteraceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Calea serrata Less. (Asteraceae), an endemic species of south Brazil known as quebra-tudo, is used in Afro-Brazilian religious rituals and in folk medicine for treating liver disorders. Phytochemical studies of the n-hexane extract of this plant demonstrated the presence of precocene II, a benzopyran derivative known for its insecticidal activity. The aim of this work was to isolate this benzopyran and determine the chemical composition of the essential oil of C. serrata and further to evaluate the acaricidal activity of the essential oil and precocene II against the larvae of Rhipicephalus (Boophilus) microplus. The LC(99.9) and LC(50) values obtained with the oil, which presents precocene II and sesquiterpenes, were 3.94 μL/mL and 0.28 μL/mL, respectively. For precocene II this values were 4.25mg/mL and 1.78 mg/mL, respectively. The results indicate a synergistic interaction between the components of the oil and precocene II.

3′-R/S-Hydroxyvoacamine, a potent acetylcholinesterase inhibitor from Tabernaemontana divaricata

Guided by the acetylcholinesterase inhibiting activity, the bisindole alkaloid 3-R/S-hydroxyvoacamine was isolated from a stem extract of Tabernaemontana divaricata, a plant used in Thailand in traditional rejuvenation remedies for improving the memory. The structure of the alkaloid was elucidated by extensive use of NMR spectroscopy and the complete assignment of the (1)H and (13)C NMR spectra is reported. The alkaloid acted as a non-competitive inhibitor against AChE with an IC50 value of 7.00±1.99 μM. An HPLC method was developed for the quantitative analysis of the AChE inhibitor. It suggested that there was 12.4% (w/w) of 3-R/S-hydroxyvoacamine in the alkaloid enriched fraction of T. divaricata stem.

Darcyribeirine, a novel pentacyclic indole alkaloid from Rauvolfia grandiflora Mart.

Three pentacyclic indole alkaloids, one novel named darcyribeirine (1) and the known isoreserpiline (2) and isoreserpine (3), were isolated from the root bark of the Rauvolfia grandiflora Mart. The structures were established by spectroscopic data.

Iridoids from Vitex cymosa

A new iridoid, named tarumal, as well as the known iridoids viteoid II and agnuside were isolated from the leaves of Vitex cymosa and identified by spectroscopic methods.

Prenylated Coumarins, Chalcone and New Cinnamic Acid and Dihydrocinnamic Acid Derivatives from Brosimum gaudichaudii

Three new natural cinnamic acid and dihydrocinnamic acid derivatives were isolated from the roots of Brosimum gaudichaudii, in addition to fourteen known substances (ten coumarins, one chalcone, b-sitosterol, 3b-O-b-D-glucopyranosylsitosterol and b-amyrin). The structures were established by spectral data, including analysis of 2D-NMR experiments and mass spectra. All aromatic compounds have 4-hydroxy-3-prenylcinnamic acid as a common precursor. The new substances are derived from a precursor of the coumarins, which through an O-methylation lost its ability to form the lactone ring.

Distribution of coumarins in the tribe Plucheeae, genus Pterocaulon

gilsane@farmacia.ufrgs.br; 2) PPG-Botanica, ICBS, 91509-900 Porto Alegre, RS, Brazil, fax (051) 33167670; 3) Grupo Metabolomica, LCQUI/CCT, Universidade Estadual do Norte Fluminense, 28015-620 Campos dos Goytacazes, RJ, Brazil; 4) Faculte de Pharmacie, Universite de Picardie Jules Verne, 80037 Amiens, France. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 572-574, November-December, 2007. Original article submitted August 1, 2006.

Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation.

Cultures from the cyanobacterial strain Microcystis aeruginosa PCC 7806 submitted to nutrient limitation become chlorotic. When returned to nutrient rich conditions these cultures regain their green colour. The aim of this study was to verify whether the cells in these cultures could be considered resting stages allowing the survival of periods of nutrient starvation as has been reported for Synechococcus PCC 7942. The experiments with Microcystis were carried out in parallel with Synechococcus cultures to rule out the possibility that any results obtained with Microcystis were due to our particular experimental conditions. The results of the experiments with Synechococcus PCC 7942 cultures were comparable to the reported in the literature. For Microcystis PCC 7806 a different response was observed. Analysis of chlorotic Microcystis cultures by flow cytometry showed that the phenotype of the cells in the population was not homogenous: the amount of nucleic acids was about the same in all cells but only around one percent of the population emitted red autofluorescence indicating the presence of chlorophyll. Monitoring of the reversion of chlorosis by flow cytometry showed that the re-greening was most likely the result of the division of the small population of red autofluorescent cells originally present in the chlorotic cultures. This assumption was confirmed by analysing the integrity of the DNA and the membrane permeability of the cells of chlorotic cultures. Most of the DNA of these cultures was degraded and only the autofluorescent population of the chlorotic cultures showed membrane integrity. Thus, contrary to what has been reported for other cyanobacterial genera, most of the cells in chlorotic Microcystis cultures are not resting stages but dead. It is interesting to note that the red autofluorescent cells of green and chlorotic cultures obtained in double strength ASM-1 medium differ with respect to metabolism: levels of emission of red autofluorescence are higher in the cells of green cultures and the ability to convert fluorescein diacetate of these cells are heterogeneous when compared to the autofluorescent cells of chlorotic cultures. Thus, the small population of the red autofluorescent cells of chlorotic cultures are in a differentiated metabolic state that allow them to persist in conditions in which most of the population loses viability; persistent cells can be detected in chlorotic cultures maintained for more than a year.

Two New Diprenylated Coumarins from Flindersia Brayleyana

Flindersia brayleyana F. Muell. contains the diprenylated coumarins 1 and 2 , in addition to the known coumarins braylin ( 3 ) and brayleyanin ( 4 ). The structures of the new natural products were elucidated by extensive NMR analysis and mass spectra.