Christa Werner

Benzoxazinoids-cyclic hydroxamic acids, lactams and their corresponding glucosides in the genus Aphelandra (Acanthaceae).

An improved method of sample preparation and simultaneous HPLC separation was developed that allowed the separation of 2,4-dihydroxy-1,4-benzoxazine-3(4H)-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3(4H)-one (DIMBOA), 2-hydroxy-1,4-benzoxazine-3(2H)-one (HBOA), 2-hydroxy-7-methoxy-1,4-benzoxazine-3(2H)-one (HMBOA) and their corresponding glucosides as well as the benzoxazolinones BOA and MBOA. The amount and distribution of these compounds was determined in the roots of Aphelandra squarrosa and A. fuscopunctata plants. There is a significant difference in the amount and distribution of this substance class in the two species analyzed. The results are discussed in relation to their function as defence compounds and allelochemicals.

Antimicrobial activities of secondary metabolites produced by endophytic fungi from Spondias mombin.

We performed a search for bioactive compounds produced by fungal endophytes from Spondias mombin (Anacardiaceae). Culture broth extracts of Guignardia sp., Phomopsis sp. and Pestalotiopsis guepinii were separated by chromatographic methods and tested for biological activities. The crude extracts of these endophytes were tested against fourteen organisms, including actinomycetes, Gram‐negative and Gram‐positive bacteria, yeast, and filamentous fungi. All fungal extracts inhibited actinomycete growth. Guignardia sp. was also active against Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, Geotrichum sp., and Penicillium canadensis. Culture extracts ofP. guepinii were active against S. cerevisae, while strains of Phomopsis sp. showed a pronounced antifungal effect against Cladosporium elatum, Mycotypha sp. and S. cerevisae.

Di-coumaroylspermidines and tri-coumaroylspermidines in anthers of different species of the genus Aphelandra

Abstract N 1 , N 5 -Di- p -coumaroylspermidine, N 5 , N 10 -di- p -coumaroylspermidine, and N 1 , N 5 , N 10 tri- p -coumaroylspermidine were isolated from anthers of Aphelandra tetragona and A. chamissoniana . The correct substitution pattern of the di-coumaroylspermidines could be identified by their specific photoisomerization to four isomers which can be separated by HPLC.

Guignardic Acid, a Novel Type of Secondary Metabolite Produced by the Endophytic Fungus Guignardia sp.: Isolation, Structure Elucidation, and Asymmetric Synthesis

A UV-guided fractionation of the AcOEt extract of the fermentation broth of Guignardia sp., an endophytic fungus from the leaves of the tropical tree Spondias mombin, resulted in the identification of the new metabolite (−)-(2S,5Z)-2-(1-methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylic acid (1), isolated as NH salt 1a. The metabolite 1 was designated (−)-(S)-guignardic acid. This first member of a new class of natural compounds contains a dioxolanone moiety formed by fusion of 2-oxo-3-phenylpropanoic acid (phenylpyruvic acid) and 3-methyl-2-oxobutanoic acid (dimethylpyruvic acid), products of the oxidative deamination of phenylalanine and valine, respectively. The structure of 1a was deduced from spectral data (UV, IR, MS, 1H- and 13C-NMR) and confirmed by asymmetric synthesis.

Putrescine, spermidine, spermine, and related polyamine alkaloids.

Publisher Summary This chapter discusses the chemistry, biological, and pharmacological aspects of putrescine, spermidine, spermine, and related polyamine alkaloids. Aliphatic di- and polyamines are widespread in microorganisms, plants, and animals, as conjugates to a number of different biological structures, and also in free form. They are known since; Lewenhoeck examined a linear tetra-amine derivative obtained from sperm, spermine phosphate. In addition to the almost ubiquitous spermine, and the two equally abundant compounds putrescine and spermidine, a great number of other biogenic di-, tri-, and tetra-amine compounds are known. Among these, sym -nor-spermidine, homospermidine, sym-homospennidine, and sym-homospermine are of particular relevance for this treatise because they occur together with putrescine, spermidine, and spermine as fundamental building blocks in polyamine alkaloids. For pharmacological studies, two biological properties of the polyamines and their derivatives are of significance. The first concerns the influence of polyamines on cell proliferation, which makes the polyamines and their derivatives interesting compounds for investigations in the field of neoplastic diseases. The second concerns the interactions of polyamines with ion channels and related receptors, which is interesting with regard to diseases of the central nervous system. With respect to cancer research, the biosynthetic enzymes of polyamines were considered as promising targets for chemotherapy. In particular, spermidine/spermine N 1 -acetyltransferase, the highly regulated key enzyme of polyamine degradation, represents a new target for novel antineoplastic agents. Many synthetic analogs of the natural polyamines have been tested as agents against different cancers, and in some cases the results are rather promising. The interaction of polyamines with ion channels is of central importance for neurotransmittance.

Hydroxylated 2-Amino-3H-phenoxazin-3-one Derivatives as Products of 2-Hydroxy-1,4-benzoxazin-3-one (HBOA) Biotransformation by Chaetosphaeria sp., an Endophytic Fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)- amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.

Accumulation and metabolism of the spermine alkaloid aphelandrine in roots of Aphelandra tetragona

Abstract The accumulation of the spermine alkaloid aphelandrine was studied in Aphelandra tetragona roots during one growth cycle using analytical HPLC. Highest aphelandrine accumulation with 7 mg g−1 dry wt and 9 mg g−1 dry wt was observed in root tissue 16 and 26 weeks after planting. Just before flower formation, the aphelandrine content in the roots decreases indicating remobilization of the alkaloid. An enzymatic degrading system for aphelandrine was also found in root tissue. 2(3H)-Benzoxazolinone and 6-methoxy-2(3H)-benzoxazolinone are liberated when the integrity of root cells is destroyed.

Interactions between agarose-embedded plant protoplasts and germ tubes of Phytophthora

Abstract Upon establishing contact, germ tubes and hyphae of Phytophthora megasperma f.sp. glycinea either penetrate soybean protoplasts (2–4% of cases), grow along their surface (12–17% of cases) or continue their growth after contact with no directional change. No appressoria are formed prior to penetration, and during penetration the pathogen is always surrounded by a continuously expanding host plasma membrane. Contact between protoplast and pathogen frequently triggers the production of finely fibrillar material at the contact sites which stains positively with the fluorescing dye “diethanol”, indicative of polysaccharides. The material is probably produced by the host protoplast and may be similar to material formed at points of apposition in the wall during infection of the intact tissue. It is not primarily responsible for the observed physical adhesion of hyphae to protoplasts. No differences in the reaction pattern were observed between two near-isolines differing mainly in their resistance against the fungal pathovar used. However, production of the wall-like material was much less frequently observed in non-host combinations (P. infestans on soybean, P. megasperma f.sp. glycinea on barley).

Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l-1, thiamine 1 mg l-1, 2,4-D 1 mg l-1, kinetin 0.2 mg l-1 and sucrose 30 g l-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g-1 of dry wt and 2.6 μmol g-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines.

Two macrocyclic spermine alkaloids from Aphelandra fuscopunctata (Acanthaceae)

Abstract Using HPLC-UV(DAD)-MS the MeOH/H 2 O/HOAc extract of the roots of Aphelandra fuscopunctata (Acanthaceae) was analyzed. The known alkaloid (+)-aphelandrine and two new macrocyclic spermine alkaloids of the (+)-aphelandrine type were isolated, namely (+)-N(6)-hydroxy-aphelandrine and (+)-N(6)-acetoxy-aphelandrine. Their constitutions and absolute configurations were established. (+)-N(6)-Hydroxy-aphelandrine was obtained from (+)-aphelandrine and (+)-N(6)-acetoxy-aphelandrine.