Band 57, Heft 5-6

The chemical composition of a dichloromethane extract of the South American liverwort Porella recurva has been examined. Two new pinguisane-type norsesquiterpenoid natural products were isolated: 6,11-epoxy-15-nor-3,4-dioxo-5,10-pinguisadien-12-acetate and 6,11- epoxy-15-nor-4-oxo-5,10-pinguisadien-12-acetate. In addition two known pinguisane-type sesquiterpenes were also isolated: norpinguisone and norpinguisone methyl ester. All structures were elucidated by means of NMR spectroscopic techniques and mass spectrometry.

The ethyl acetate extract of roots of Tephrosia candida afforded three new spirorotenoids belonging to a new class of spirocompounds, named tephrospirolactone, tephrospiroketone I, and tephrospiroketone II. The structures of these compounds were determined mainly based on spectral analysis. The only known spirorotenoid described in the literature is amorphispironone, isolated from Amorpha fruticosa.

A new steroidal saponin was isolated from the leaves of Agave attenuata Salm-Dyck. Its structure was established as (3β,5β,22α,25S)-26-(β-ᴅ-glucopyranosyloxy)-22-methoxyfurostan- 3-yl O-β-ᴅ-glucopyranosyl-(1→2)-β-ᴅ-glucopyranosyl-(1→2)-O-[β-ᴅ-glucopyranosyl- (1→3)]-β-ᴅ-glucopyranosyl-(1→4)-β-ᴅ-galactopyranoside. The structural identification was performed using detailed analyses of 1 H and 13 C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR and COLOC) and chemical conversions. The haemolytic potential of the steroidal saponin was evaluated and the anti-inflammatory activity was performed using the capillary permeability assay.

Alkaloid profiles of two Egyptian Senecio species (Senecio aegyptius var. discoideus and S. desfontainei) in addition to a cultivated species (S. cineraria) were studied using capillary GLC and GLC-mass spectrometry with respect to pyrrolizidine alkaloids (PAs). Four alkaloids were identified in S. aegyptius var. discoideus, 8 in S. desfontainei and 13 in S. cineraria. Some of these alkaloids have not been reported from these plants. The alkaloidal pattern of different plant organs (flowers, leaves, stem, root) were also investigated. Senecionine has been found to be a one of the major alkaloid in all studied species, it was isolated and its structure was elucidated by 1 H- and 13 C-NMR.

The essential oil of Senecio aegyptius var. discoideus flowers, leaves, stems and roots were isolated by hydrodistillation. Analysis of the oils by capillary GLC and GLC-mass spectrometry were performed and 34 out of 37 compounds were identified. The main component was isolated and characterized as 1,10-epoxyfuranoeremophilane using a combination of GLC, GLC-MS, and NMR analyses. The oils of flowers, leaves and stems were rich in monoterpene hydrocarbons while the root oil mainly contains furanoeremophilanes. Flower and leaf volatile oils showed significant level of antifungal activity against C. albicans, moderate effect against Grampositive bacteria, however, it has weak activity against Gram negative bacteria. The isolated sesquiterpene (1,10-epoxyfuranoeremophilane) exhibited substantial inhibitory activity against Gramnegative bacteria.

A phytochemical investigation of the flowers and leaves of Ficaria verna Huds. (Ranunculaceae) yielded four additional known flavonoid compounds including: kaempferol 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-β-ᴅ-glucopyranoside (vitexin), luteolin 8-C-β-ᴅ-glucopyranoside (orientin) and apigenin 8-C-β-ᴅ-(2ʺ-O-β-ᴅ-glucopyranosyl)- glucopyranoside (flavosativaside). The characterisation of these compounds was achieved by various chromatographic and spectroscopic methods (UV, 1 H NMR, 13 C NMR and MS).

Externally accumulated flavonoid aglycones have been found for the first time in Nonea species. They exhibit only flavones, one of thembeing the rare tricetin-4ʹ-methyl ether. Within the subfamily Boraginoideae, exudate flavonoids appear to be a rare character.

The essential oil of the leaves of P. racemosa var. terebinthina and P. racemosa var. grisea were examined by GC and GC/MS. The major constituents were α-terpineol acetate (27%), α-terpineol (20%) and 4-methoxy eugenol (12.6%) for P.racemosa var. terebinthina and 4-methoxy-isoeugenol (75.2%) and 4-methoxy-eugenol (4.5%) for P. racemosa var. grisea.

Extracellular polysaccharide mixture that forms a viscous mucilate in the fermentation medium during industrial cultivation of Penicillium vermiculatum was isolated by ethanol precipitation and its structural characteristics were investigated by a combination of physicochemical methods. The mixture contained two structurally different polysaccharides similar to those previously described for some fungal species. This is the first report of the fully structurally characterized extracellular polysaccharides of Penicillium vermiculatum.

Aspterric acid (1) and 6-hydroxymellein (2), inhibitors of pollen development in Arabidopsis thaliana, have been isolated fromthe fungus Aspergillus terreus. 1 and 2 inhibited the pollen development at concentrations of 38 and 52 μᴍ, respectively. The microscopic examination of pollen development suggested that the inhibition by the treatment with 1 caused at meiosis and the inhibition by the treatment with 2 caused at microspore stage. 1 and 2 could be useful agents for the molecular investigation of anther and pollen development in higher plants.

The dual culture of Trichoderma harzianum and Catharanthus roseus callus produced an antimicrobial compound with a remarkable activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. Structural elucidation revealed that this compound, which we have named trichosetin, is a novel tetramic acid (2,4-pyrrolidinedione) antibiotic and a homolog of the fungal metabolite equisetin. This compound however, was not produced in the individual culture of T. harzianum or C. roseus callus.

Mycosporine like amino acids (MAAs) were detected in low concentration in sea ice algae growing in situ at Cape Evans, Antarctica. Four areas of sea ice were covered with plastics of different UV absorption exposing the bottom- ice algal community to a range of UV doses for a period of 15 days. Algae were exposed to visible radiation only; visible + UV radiation; and visible + enhanced UV radiation. MAA content per cell at the start of the experiment was low in snow-covered plots but higher in samples from ice with no snow cover. During the study period, the MAA content per cell reduced in all treatments, but the rate of this decline was less under both ambient UV and visible radiation than under snow covered plots. While low doses of UVB radiation may have stimulated some MAA production (or at least slowed its loss), relatively high doses of UVB radiation resulted in almost complete loss of MAAs fromice algal cells. Despite this reduction in MAA content per cell, the diatoms in all samples grew well, and there was no discernible effect on viability. This suggests that MAAs may play a minor role as photoprotectants in sea ice algae. The unique structure of the bottom ice algal community may provide a self-shading effect such that algal cells closest to the surface of the ice contain more MAAs than those below them and confer a degree of protection on the community as a whole.

The chemical composition of the essential oils from the needles of Pinus ponderosa (north american pine), P. resinosa (red pine) and P. strobus (eastern white pine) has been determined by GC/MS (FID). The essential oils from P. resinosa and P. ponderosa in comparison to P. strobus have been characterized by the higher content of β-pinene (42.4%, 45.7% and 7.9% respectively). On the other hand, α-pinene (17.7%) and germacrene D (12.2%) were dominant compounds of P. strobus. Moreover the essential oil from P. resinosa was more rich in myrcene-15.9%. Estragole and Δ-3-carene, each one in amount ca 8% were identified only in P. ponderosa. The content of essential oils in the needles slightly varied - 0.65% - P. resinosa, 0.4% - P. strobus, 0.3% - P. ponderosa. The antifungal activity has been investigated towards Fusarium culmorum, F. solani and F. poae. The strongest activity was observed for the essential oil from P. ponderosa, which fully inhibited the growth of fungi at the following concentrations - F. culmorum, F. solani at 2% and F. poae at 5%.

A mixture of flavalignan cinchonains Ia and Ib was isolated from the bark of Trichilia catigua. The structures were established on the basis of spectroscopic data of the natural products and their methylated derivatives including 2D NMR experiments, and compared with data in the literature. These flavalignans exhibited antibacterial activity against Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

The biotransformation of a mixture of argentatin A (20%) 1 and incanilin (80%) 2 by Gibberella suabinetti ATCC 20193 and Septomyxa affinis ATCC 6737 demonstrated the conversion of incanilin to 16β-hydroxylanosta-2, 8, 23-triene, while argentatin A did not react. The acetate of this triterpenoid mixture was biotransformed by Septomyxa affinis ATCC 6737 to give five metabolites. Argentatin A acetate was transformed to 3β, 16β,30-trihydroxycycloart- 20, 24-diene, 20R, 24R-epoxy-16β, 25-dihydroxy-3, 4-seco-cycloart-4(28)-en-3-oic acid acetate and 20R, 24R-epoxy-16β, 25-dihydroxy-3, 4-seco-cycloart-4(28)-en-3-oic acid. Incanilin acetate was converted to 16β-hydroxylanosta-2, 8, 23-triene and 20R, 24R-epoxy-16β, 25-dihydroxy-3, 4-seco-lanost-1, 4(28), 8-trien-3-oic acid acetate. The structural elucidations of these metabolites were achieved by different spectroscopic methods.

The activity of every substance I inhibiting an enzymatic reaction can be approximately evaluated by the index pI 50 . This paper describes a simple and fast method of estimate and/or determination of this index. The method is based on the linearity of the dependence of the ratio of reaction rates of uninhibited and inhibited reaction vs. concentration of the inhibitor at constant initial substrate and enzyme concentrations for fully competitive, noncompetitive, uncompetitive and mixed type of inhibition by the one inhibitor. The validity of the method is demonstrated by four inhibitors of hydrolysis of acetylthiocholine by butyrylcholine esterase.

In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 ± 0.47 nmol Pi/h x 10 8 cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent K m for p-NPP was 5.35 ± 0.89 mᴍ. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl 2 and sodiumfluoride , as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.

The aim of this work was to evaluate, in vitro, the effect of ʟ-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by ʟ-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mm of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 μᴍ substrate, p<0.01). Lineweaver-Burk plots showed that V max remained unchanged. However, K M was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.

A study was performed on the oxidizing degradation of 2-chloroaniline (used as a model pollutant in water) by photolysis (λ = 254 nm). The change of spectrum and substrate concentration of treated solutions was measured spectrophotometrically as well as by HPLC. The yields of the degradation products (chloride ions, ammonium ions, formaldehyde, etc.) were studied as a function of UV-dose. Their initial quantumyields (Q i ) were determined by specific analysis. It was shown that the substrate photolysis in the presence of N 2 O is most efficient, followed by degradation in media saturated with pure oxygen and air. A probable reaction mechanism for the photo-induced degradation of 2-ClA is presented.

The genetic diversity of 33 Nigerian Helicobacter pylori isolates were studied using RAPD, PCR-RFLP and Southern blot analysis of ureA or ureCD gene probes. RAPD was able to distinguish the following number of isolates using the primers 3880 : 5ʹ-AAGAGCCCGT-3ʹ (28), 3881 :5ʹ-AACGCGCAAC-3ʹ (33) and OPH8 :5ʹ-GAAACACCCC-3ʹ (25). Southern blot analysis using the ureCD probe was also able to distinguish the 12 isolates tested into ten different patterns. The PCR-RFLP technique distinguished all 33 isolates into six types. In conclusion, considering typeability, discriminatory power, and convenience, RAPD with the 3881 primer was considered the most useful technique.

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen- rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.

Ten propolis samples from Bulgaria, Italy and Switzerland were analyzed by GC-MS. As expected, most samples displayed the typical chemical pattern of “poplar” propolis: they contained pinocembrin, pinobanksin and its 3-O-acetate, chrysin, galangin, prenyl esters of caffeic and ferulic acids. Two samples differed significantly: one from the Graubünden Alpine region, Switzerland, rich in phenolic glycerides, and one from Sicily which contained only a limited number of phenolics and was rich in diterpenic acids.

Sterols, volatiles and lipids were isolated and identified from lipophylic extracts from two tunicates, Styela sp. and Phallusia sp., occurring in the Eastern Mediterranean. Seventeen sterols were identified. The sterol composition of the two organisms appeared to be similar except for the concentrations of 5α-stanols. Both tunicates were characterized by the presence of sterols with a (22Z)-double bond. In the volatiles significant amounts of chlorinated compounds were found (phenols in Styela sp. and hydrocarbons in Phallusia sp.). The fatty acid composition of triacylglycerols and phospholipids of the two tunicates showed significant differences.

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO 2 )-Phe-Val-Leu-O 4 MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-β-PMA efficiently inhibited the activity of the 68-kDa protease.

The marine alkaloids haminol A, haminol B and pulo’upone as well as 17 related compounds (twelve 2-substituted pyridine derivatives, four 3-substituted ones and one analogue of the bicyclic terminus of pulo’upone) were tested for antimicrobial activity against a panel of six microbes (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans and Saccharomyces cerevisiae) using the paper disc agar diffusion method. Six compounds were tested also against the mold Aspergillus niger. Some of the compounds displayed noteworthy antimicrobial activity, only one congener being completely devoid of activity. Nearly all compounds had activity against B. cereus and S. epidermidis. The growth of E. coli, C. albicans and S. cerevisiae was also distinctly inhibited by many compounds. In contrast, most compounds were inactive or had minimal activity against P. aeruginosa. Interestingly, most of the compounds tested against the opportunistic pathogen A. niger were active, one of them having noteworthy inhibitory potency.

Two male-specific beetle volatiles were found that elicited strong gas chromatographic-electroantennographic responses from both sexes of Asian longhorned beetle adults, Anoplophora glabripennis. The secretion consisted of a ~1:1 (v/v) blend of functionalized dialkyl ethers, 4-(n-heptyloxy)butanal and 4-(n-heptyloxy)butan-1-ol. These compounds are chemically unusual natural products that are previously unknown frominsects . Laboratory olfactometer studies showed that a blend of 10 μg of each synthetic compound on a filter paper strip was significantly attractive to ALB adults.

The alkaloid spectrumin roots, leaves and seeds of Datura stramonium L. was investigated by GC-MS. Twenty-nine tropane alkaloids are detected. Twelve of themare new constituents for the species and the two tropane esters 3-(3ʹ-acetoxytropoyloxy)tropane (21) and 3-(2ʹ-hydroxytropoyloxy)tropane (26) are described for the first time.

The method describes a simultaneous determination of inorganic phosphate (Pi) and protein content from a reaction mixture used for assay of adult rat cerebrocortical synaptosomal membrane Na + -K + -ATPase specific activity. The present method is more convenient, accurate and quicker compared to the existing methods for the determination of Na + -K + -ATPase activity. It also eliminates the possible errors in protein estimation by other classical methods in brain, which have a high lipid content.