Band 57, Heft 7-8

A new red anthraquinone, draculone, has been isolated from the corticolous tropical lichen Melanotheca cruenta (= Trypethelium cruentum = Pyrenula cruenta) together with minor quantities of the known anthraquinone pigment haematommone. The structure of draculone was determined as 2-acetyl-1,3,4,6,8-pentahydroxyanthraquinone by spectroscopic methods.

Aerial parts of Achillea collina afforded, in addition to five known sesquiterpene lactones two new germacranolides and one new eudesmanolide, the structures of which were elucidated by spectroscopic methods.

A new sesquiterpene of humulene type, named 2β, α-epoxy-6Z, 9Z-humuladien-8α-ol (1) together with a known compound lactarinic acid was isolated from the fruiting bodies of Lactarius hirtipes. Their structures were established on the basis of spectral evidence (MS, IR, NMR, HMBC, HMQC, 1 H- 1 H COSY, ROSEY experiments). Compound 1 was found as the first humulene-type sesquiterpene in higher fungi.

Two new oleanane-type triterpenes, characterized as 3-oxo-11α,12α-epoxy-oleanan-28,13β- olide and 3-oxo-olean-11-en-28,13β-olide , were isolated from the fruits and seeds of Cedrela montana (Meliaceae). In addition, the known compounds oleanonic acid , a mixture of β-sitosterol and stigmasterol, and the limonoid photogedunin were also isolated. The structures of the new compounds were established by spectroscopic methods, including 2D NMR.

The roots and aerial parts of Tephrosia major Micheli, afforded a new prenylated-β-hydroxychalcone, characterized as 2′,6′-dihydroxy-3′-prenyl-4′-methoxy-β-hydroxychalcone. In addition, seven prenylated flavonoids, two rotenoids, β-sitosterol, stigmasterol, lupeol and quercetin were isolated. The structure of the new β-hydroxy chalcone was established by spectroscopic methods, including 2D NMR experiments.

The chemical composition of the brown alga Cystoseira crinita Bory from the Eastern Mediterranean was investigated. Fourteen sterols have been identified, five of them for the first time in algae. The structure of one new sterol was established. The origin of seven sterols with short side chains was discussed. In the volatile fraction 19 compounds and in the polar fraction 15 compounds were identified. The main lipid classes were isolated and their fatty acid composition was established.

From the methanolic extract of the underground parts of Globularia orientalis, a new antioxidant sugar ester was isolated. The structure of the new compound, globularitol (1), was identified as 6-O-feruloyl-β-ᴅ-glucopyranosyl-(156)-glucitol by spectroscopic methods (1D and 2D NMR, ESI- and FAB-MS) and confirmed by chemical means.

The new triterpene saponin 3-O-β-ᴅ-glucopyranoside, 28-β-ᴅ-glucopyranosyl-(1→2)-β-d-glucopyranosiduronic acid oleanolate was isolated from the roots of Chenopodium ficifolium. The known compounds stigmasterol-3-O-glucoside and 3-O-β-ᴅ-glucopyranosiduronic acid, 28-β-ᴅ-glucopyranosyl oleanolate were also isolated. The latter compound, oleanolic acid, β-sitosterol and its glucoside were isolated from the aerial parts. The identity of these compounds was verified through different chemical and physico-chemical evidences including different 1D and 2D NMR experiments.

A new nuatigenin-type steroidal saponin, multifidoside (2), was isolated from the aerial parts of Veronica fuhsii and V. multifida and its structure was identified as 3-O-{[α-ʟ-rhamnopyranosyl-( 1→2 glu )]-[β-ᴅ-glucopyranosyl-(1→4 rha )-α-ʟ-rhamnopyranosyl (1→4 glu )]-β-ᴅ-glucopyranosyl}nuatigenin 26-O-β-ᴅ-glucopyranoside. Additionally, a known steroidal saponoside, aculeatiside A (1), from V. fuhsii, a phenylethanoid glycoside, verpectoside A (3), and a flavon glycoside, isoscutellarein 7-O-(2″-O-6″-O-acetyl-β-ᴅ-allopyranosyl-β-ᴅ-glucopyranoside) (4) from V. multifida were isolated.

A new naturally occurring isoflavone, derrone, was isolated from Ulex jussiaei (Leguminosae) together with the isoflavones ulexins A-C, lupalbigenin, isolupalbigenin, 7-O-methylisolupalbigenin, isoderrone, ulexone A and isochandalone, the pterocarpans (6aR,11aR)-(-)- maackiain, (6aR,11aR)-(-)-2-methoxymaackiain and (6aR,11aR)-(-)-4-methoxymaackiain, the chalcone 4-hydroxylonchocarpine and the dihydrochalcone crotaramosmine. The antifungal activity of the new compound was tested by a bioautographic method against Cladosporium cucumerinum, and as expected from structural features it proved to have no activity.

A novel tyrolobibenzyl derivative, 1→6-β-ᴅ-apiosyl-β-ᴅ-glucopyranosyl 4-[2-(4-hydroxyphenyl) ethyl]benzofuran-2-carboxylate 3 (tyrolobibenzyl D) was isolated from Scorzonera humilis L. and its structure established by mass spectrometry and 1D- and 2D-NMR spectroscopy. The biological activities of the new compound and related tyrolobibenzyls A-C (1-2, 4) and the semi-synthetic peracetyl derivatives of tyrolobibenzyls B (2a) and C (4a) were assessed. The results revealed no cytotoxic activity against P388 cells and neither anti-bacterial activity against Bacillus subtilis nor antifungal activity against Candida albicans and Trichophyton mentagrophytes for any of the investigated compounds. An evaluation of potential chemopreventive activity of 2, 2a, 4, and 4a also revealed no pronounced activity in any of the employed assaying systems.

The essential oil of the aerial parts of Tanacetum santolinoides was analyzed by capillary GLC and GLC-MS. Altogether 30 components were identified. The main constituents were thymol (18%), trans-thujone (17.5%), trans-chrysanthenyl acetate (13.2%), cis-chrysanthenyl acetate (9.2%), umbellulone (9.7%) and 1,8-cineole (4.7%). Similar essential oil pattern in addition to palmitic acid methyl ester, palmitic acid, stigmasterol, sitosterol and two flavonoidal aglycons were found in the n-hexane-ether extract. The oil showed strong in vitro activity against E. coli, Bacillus subtilis and Candida albicans.

Fourteen aromatic and 24 aliphatic acids were determined by GC-MS analysis of acidic fractions obtained from Paeonia peregrina and Paeonia tenuifolia roots. Benzoic acid and its monohydroxy-, dihydroxy- and trihydroxyderivatives are the main acid components of both Paeonia species. Some fractions could serve as a source of benzoic, 4-hydroxybenzoic, vanillic and gallic acids, as well as of ethyl gallate. The fractions inhibited the growth of Staphylococcus aureus, Escherichia coli and Candida albicans.

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO 4 ·5H 2 O, CoCl 2 ·6H 2 O and FeSO 4 ·7H 2 O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.

Toxicity and accumulation of Cd 2+ in yeasts were studied in eight different yeast species. The adaptation to toxic concentration of this metal was dependent on the production of extracellular yeast glycoproteins. The highest concentration of Cd 2+ ions in the growth medium was tolerated by a Hansenula anomala, strain while the lowest tolerance was found by the strain of species Saccharomyces cerevisiae. Extracellular glycoproteins of Hansenula anomala absorbed nearly 90% of the total content of Cd 2+ ions bound by yeast cells, while extracellular glycoproteins of Saccharomyces cerevisiae bound only 6% of the total amount of cadmium. This difference is caused by the variable composition of the saccharide moiety in the extracellular glycoproteins. The composition of extracellular glycoproteins changed during the adaptation of the yeast cells to the presence of Cd 2+ ions.

Betalains biosynthesis and antiradical scavenging activity were investigated during cultivation of four hairy root cultures of Beta vulgaris, obtained from different cultivars (Bordo, Egyptian, Detroit 2 and Detroit Dark Red). The best producer of betalains was a hairy root culture from Beta vulgaris cv. Detroit Dark Red (13.27 mg/g dry weight total pigment production). The ethanol extract, derived from roots of the same culture grown for 15 days under submerged conditions, showed a high antiradical activity (83% of inhibition of the stable DPPH·).

An isolate of Fusarium solani, NRRL 18883, produces the natural phytotoxin 2,5-anhydro-ᴅ-glucitol (AhG). This fungal metabolite inhibited the growth of roots (I 50 of 1.6 mᴍ), butit did nothave any in vitro inhibitory activity. The mechanism of action of AhG requires enzymatic phosphorylation by plant glycolytic kinases to yield AhG-1,6-bisphosphate (AhG-1,6- bisP), an inhibitor of Fru-1,6-bisP aldolase. AhG-1,6-bisP had an I 50 value of 570 μᴍ on aldolase activity, and it competed with Fru-1,6-bisP for the catalytic site on the enzyme, with a K i value of 103 μᴍ. The hydroxyl group on the anomeric carbon of Fru-1,6-bisP is required for the formation of an essential covalent bond to ζ amino functionality of lysine 225. The absence of this hydroxyl group on AhG-1,6-bisP prevents the normal catalytic function of aldolase. Nonetheless, modeling of the binding of AhG-1,6-bisP to the catalytic pocket shows that the inhibitor interacts with the amino acid residues of the binding site in a manner similar to that of Fru-1,6-bisP. The ability of F. solani to produce a fructose analog that is bioactivated by enzymes of the host plant in order to inhibit a major metabolic pathway illustrates the intricate biochemical processes involved in plant-pathogen interactions.

Beta- and gamma-eudesmols mixture was microbiologically transformed by Gibberella suabinetti ATCC 20193. Seven different eudesmanoidal metabolites (3-9) were isolated and their structures were elucidated by the different spectroscopic techniques. These metabolites are: eudesma-4-en-11-ol-3-one (carissone), eudesma-3-en-2β, 11-diol, eudesma-4-en-3β,11-diol, eudesma-4(15)-en-8,11-diol, eudesma-4(15)-en-2α,11-diol (pterocarpol), 1(3)cyclo-eudesma-4(15)-en-11,12-diol and eudesma-4-en-11,15-diol.

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, 1 H, 13 C NMR and 2D NMR techniques and confirmed by chemical transformation.

Five strains of filamentous fungi (Aspergillus niger strains NRRL 3122 and T0005007-2, Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in ζ-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against ζ-carotene desaturase inhibitors.

The biotransformation of partheniol by the fungus Calonectria decora ATCC 14767, produced six metabolites. These metabolites are; aromadendr-1(10)-en-8α -ol; 5(9),6-tricyclohumul- 1(10)-en-8α-ol; 4,15-didehydro-6-bicyclohumulan-8α,10α-diol; 5(9),6-tricyclohumulan- 4α,8α-diol; maalian-1α,8α-diol and 14-epi-1(4)-epoxymaalian-8α-ol. The identities of these metabolites were established by different spectroscopic measurements.

(-)-β-pinene, a flavor and fragrance monoterpene is an important constituent of essential oils of many aromatic plants. It was oxidized by a plant-pathogenic fungus, Botrytis cinerea to afford four metabolites characterized as (-)-6α-hydroxy-β-pinene, (-)-4β,5β-dihydroxy- β-pinene, (-)-2β,3β-dihydroxypinane, and (-)-4β-hydroxy-β-pinene-6-one by detailed spectroscopic studies along with other known metabolites.

Trying to detect the genes coding for light harvesting II polypeptides of the purple bacteria Ectothiorhodospira sp. a gene corresponding to a light harvesting I polypeptide was cloned. This paper discusses the probable reasons of this result. The sequence of this polypeptide underlines the strong similarity with light harvesting complexes from bacteria such as Rhodospirillum molischianum and Chromatium vinosum.

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2·10 -5 ᴍCa 2+ . Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.

Glucose oxidase (E. C. 1.1.3.4) was immobilized on electrochemically modified graphite to obtain an enzyme electrode. The working surface of the electrode was coated with gelatine to prevent desorption of the enzyme. In substrate (glucose) solutions the amperometric signal of the enzyme electrode was due to the electroreduction of H 2 O 2 generated in the enzyme layer. The linearity of the electrode response was found up to a substrate concentration of 300 μᴍ at a working potential of 0 mV (vs. Ag/AgCl). It was shown that the electrode did not respond to l-ascorbic and uric acid at that working potential. The response time was about 2 min. The enzyme electrode keeps about 50% of its initial activity after a one-week storage at 4 °C.

The effect of three anions, Cl - , Br - and I - , on the phase transitions of dipalmitoylphosphatidylcholine (DPPC) was measured. Main phase transition was modestly affected by these anions in the salt concentration range 0.2 M. For Cl - and Br - the temperature of main phase transition was lower (by about 0.5 °C), its half-width modestly larger and enthalpy practically unchanged, all three parameters were altered to a much larger degree. Main phase transition temperature was 1.5 °C lower and the peak half-width significantly smaller. These changes were not accompanied by any alteration in main phase transition enthalpy. Iodide shifted the pretransition temperature toward lower values and increased its half-width to such an extent that at concentrations above 100 mm it was practically undetectable. Besides cations, the presence of anions also has a distinct effect on lipid bilayer interface properties.

A series of aurones was analyzed for the ability to inhibit respiratory functions of mitochondria of Leishmania parasites. The aim of this study was to find a rational explanation for the activity of certain aurones and auronols as novel antiprotozoal compounds of plant origin. In a cell-free assay mitochondrial fumarate reductase from L. donovani was inhibited in a concentration-dependent manner. The most active compounds were 4′,6-dihydroxyaurone and 6-methoxyaurone which inhibited parasite enzyme activity at 25 nᴍ by over ninety percent.

High temperature high resolution gas chromatography coupled to mass spectrometry (HTHRGC- MS) isa powerful analytical tool. In this work we applied this technique to the study of crude extracts of propolis collected near the city of Uberlândia - Minas Gerais State. Eucalyptus trees and native plants from “cerrado” (savannah) were the material sources disposable for the Apis mellifera bees. A lot of known propolis constituents were identified, however, several high molecular weight compounds including lupeol alkanoates were identified for first time in propolis.

The characterization of metallothionein in the Urodele amphibian species Pleurodeles waltl was achieved. A simple and rapid method for identification of metallothionein, based on its strong affinity for cadmium ( 109 Cd), was used. We were able to show that metallothionein is constitutively synthesized in liver, ovary and brain. The property of metallothionein to strongly bind essential (Zn, Cu) as well as toxic (Cd, Hg) metals is consistent with a dual role in cellular metabolism, i.e. homeostatis and detoxification of heavy metal ions.

Reexamination of the marine sponge Suberea aff. praetensa, (Row) from the Gulf of Thailand furnished in addition to bromotyrosine derivatives found previously 5-bromo- and 5- chlorocavernicolin, cavernicolins 1 and 2, two other brominated tyrosine metabolites, a known bisoxazolidone and a new unusual rearranged tyrosine metabolite subereatensin. Several of the metabolites exhibited significant inhibitory effects against five human cancer cell lines.

Mass spectrometric investigations confirmed the structure of the female produced sex pheromone of the horse-chestnut leafminer Cameraria ohridella Desch. and Dim. to be (8E,10Z)-8,10-tetradecadienal. Pure samples, prepared in a straightforward synthesis, were highly attractive in field tests and proved to be suitable for monitoring of flight activities and population dynamics. In mixtures with the synthetic pheromone, analogues like 9-tridecynal and 7-dodecynyl formate were shown to reduce trap catches. In electroantennographic experiments, pheromone analogues were less active than the pheromone. 9-Tridecynal was the most EAG active analogue tested, followed by 7-dodecyn-1-yl formate and 7-undecyn-1- yl formate.

Virgin female gland extracts of sugarcane moth Diatraea saccharalis (Fabricius) (Lepidoptera: Pyralidae), from three locations in Brazil, have been analyzed. By GC-MS analysis and comparison of the chromatographic retention time of the components of the pheromone gland with those retention times of synthetic standards, we observed the presence of (Z)- hexadec-11-enal (1), hexadecanal (2), (9E,11Z)-hexadecadienal (4), (9Z,11Z)-hexadecadienal (5) and (9E,11E)-hexadecadienal (6), as minor components besides the major constituent (9Z,11E)-hexadecadienal (3) already reported. We found no variations in the composition of the gland extracts deriving from the three Brazilian populations and only two compounds, (Z)-hexadec-11-enal (1) and (9Z,11E)-hexadecadienal (3), elicited antennal responses (GCEAD). In electroantennography (EAG), however, pure compounds 1 and 3, a binary mixture containing 1 and 3, and a mixture containing all of the six synthetic compounds 1-6 elicited a depolarization in male antennae of D. saccharalis, without any statistically different delay. The EAG responses to the other isomers of 9,11-hexadecadienal were small and not significantly different from the control, except for the (9Z,11Z)-isomer (5) which showed an relatively strong electroantennal activity.

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, is a widely used model system to study the cellular and molecular events involved in differentiation of leukemic cells. In a screening for inducers of differentiation of HL-60 cells, cultures of Gliocladium strain 4-93 were found to produce Sch 52900, a previously isolated diketopiperazine (Chu et al,. J. Antibiotics 48, 1440-1445). Sch 52900 induced the differentation of 50-69% of HL-60 cells at concentrations of 6.8-13.6 nᴍ as measured by nitro-blue tetrazolium chloride (NBT) reduction which was followed by apoptosis as shown by DNA fragmentation. Our results demonstrate that growth arrest and the induction of differentiation by Sch 52900 is due to the induction of the cell cycle inhibitor p21 WAF and an inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway which leads to the activation of the transcription factor AP-1.