Volume 61, Issue 7-8

A total of 23 volatile constituents was identified and characterized by GC and GC-MS in the volatile essential oil extracted from intact (juvenile and adult) and fallen (senescent and leaf litter) leaves of lemon-scented eucalyptus (Eucalyptus citriodora Hook.). The leaves differed in their pigment, water and protein content, and C/N ratio. The oils were, in general, monoterpenoid in nature with 18 monoterpenes and 5 sesquiterpenes. However, a great variability in the amount of essential oils and their individual constituents was observed in different leaf tissues. The amount was maximum in the senescent leaves collected from the floor of the tree closely followed by that from juvenile leaves. In all, 19 constituents were identified in oil from juvenile and senescent leaves compared to 23 in adult leaves and 20 in leaf litter, respectively. Citronellal, a characteristic monoterpene of the oil reported hitherto was found to be more (77D78%) in the juvenile and senescent leaves compared to 48 and 54%, respectively, in the adult leaves and leaf litter. In the adult leaves, however, the content of citronellol - another important monoterpene - was very high (21.9%) compared to other leaf types (7.8-12.2%). Essential oil and its two major monoterpenes viz. citronellal and citronellol were tested for their phytotoxicity against two weeds (Amaranthus viridis and Echinochloa crus-galli) and two crops (Triticum aestivum and Oryza sativa) under laboratory conditions. A difference in the phytotoxicity, measured in terms of seedling length and dry weight, of oil from different leaves and major monoterpenes was observed. Oil from adult leaves was found to be most phytotoxic although it occurs in smaller amount (on unit weight basis). The different toxicity of different oil types was due to the relative amount of individual monoterpenes present in the oil, their solubility and interactive action. The study concludes that oil from senescent and juvenile leaves being rich in citronellal could be used as commercial source of citronellal whereas that from adult leaves for weed management programmes as it was the most phytotoxic.

(-)-Agelasidine A was identified from the methanol extract of the marine sponge Agelas clathrodes for the first time together with zooanemonin, 1-carboxymethylnicotinic acid, hymenidin, mukanadins A and C, monobromodispacamide, agelasidine D, 2-amide-4-bromopyrrole, O-methyltryptophan and an agelasines mixture. The structures were characterized by spectroscopic methods. (-)-Agelasidine A was tested for antibacterial and antifungal activities and shown to act as a bacteriostatic agent as it inhibited the growth of Staphylococcus aureus and partially the growth of other bacteria.

The aim of this work was to use in vivo models to evaluate the analgesic and anti-inflammatory activities of ursolic acid (UA) and oleanoic acid (OA), the major compounds isolated as an isomeric mixture from the crude methylene chloride extract of Miconia albicans aerial parts, in an attempt to clarify if these compounds are responsible for the analgesic properties displayed by this plant. Ursolic acid inhibited abdominal constriction in a dose-dependent manner, and the result obtained at a content of 40 mg kg -1 was similar to that produced by administration of acetylsalicylic acid at a content of 100 mg kg -1 . Both acids reduced the number of paw licks in the second phase of the formalin test, and both of them displayed a significant anti-inflammatory effect at a content of 40 mg kg -1 . It is noteworthy that the administration of the isolated mixture, containing 65% ursolic acid/35% oleanolic acid, did not display significant analgesic and anti-inflammatory activities. On the basis of the obtained results, considering that the mixture of UA and OA was poorly active, it is suggested that other compounds, rather than UA and OA, should be responsible for the evaluated activities in the crude extract, since the crude extract samples displayed good activities.

Four diterpenoids, ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone, were isolated from transformed roots of Salvia sclarea. Salvipisone and aethiopinone showed relatively high cytotoxicity against HL-60 and NALM-6 leukemia cells (IC 50 range 0.6-7.7 μg/ mL which is equal to 2.0-24.7 μᴍ), whereas 1-oxoaethiopinone and ferruginol were less active in this regard. Moreover, we have found that all four diterpenoids of S. sclarea had equal cytotoxic activity against parental HL-60 and multidrug-resistant HL-60 ADR cells, what indicates that they are poor substrates for transport by multidrug resistance-associated protein (MRP1). Caspase-3 activity determinations showed that salvipisone and aethiopinone were able to induce apoptosis in a time- and concentration-dependent manner. The results obtained in this study show that S. sclarea diterpenoids aethiopinone and salvipisone may be useful in the treatment of human cancers, especially in the case of drug resistance.

Coleonema album, a member of the South African fynbos biome, was evaluated for its antimicrobial activity associated with its secondary metabolites. Ethanol- and acetone-based extracts obtained from plants from two different geographical areas were analyzed. A bioassay- guided fractionation methodology was followed for rapid and effective screening for the presence of bioactive compounds. The TLC-bioautographic method, used to screen the plant extracts for antimicrobial activity and localization of the active compounds, indicated the presence of a number of inhibitory compounds with activity against the microorganisms (E. coli, B. subtilis, E. faecalis, P. aeruginosa, S. aureus, M. smegmatis, M. tuberculosis, C. albicans, C. cucumerinum) tested. Evaluation of the inhibitory strength of each extract by the serial microdilution assay indicated that the C. album extracts inhibited effectively all the microorganisms, with the minimum inhibitory concentrations in the low mg ml -1 range. Identification and structural information of the bioactive components were obtained by a combination of preparative TLC and LC-MS. It revealed the presence of coumarin aglycones which were responsible for the observed antimicrobial activities. The results of this study indicate that C. album possesses strong antimicrobial activity against a wide range of microorganisms that warrants further investigation into the use of the extracts or their active constituents as a potential source for novel drugs.

The antimicrobial activity and the MIC values of the ethanol, chloroform, diethyl ether, and acetone extracts of the chemical races of Pseudevernia furfuracea (var. furfuracea and var. ceratea) and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents have been investigated against some microorganisms. Nearly all extracts of both chemical races showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, Candida glabrata, Alternaria alternata, Ascochyta rabiei, Aspergillus niger, Fusarium culmorum, Fusarium moniliforme, Fusarium oxysporum, Fusarium solani, and Penicillium notatum. There was no antimicrobial activity of the extracts against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella typhimurium, Alternaria citri, Alternaria tenuissima, and Gaeumannomyces graminis. Chloroatranorin and olivetoric acid were active against the same microorganisms with few exceptions. Physodic acid was active against about the same bacteria and yeasts and inactive against all of the filamentous fungi tested. Also no activity of atranorin against the filamentous fungi was observed.

From the aerial roots of the medicinal plant Geranium sanguineum L. a polyphenol-rich extract with strong anti-influenza activity has been isolated. To investigate its active fractions, the extract was partitioned by solvents with increasing polarity. The n-BuOH fraction contained the majority of the in vitro antiviral activity; the EtOAc fraction was the most effective one in vivo. A bioassay-directed fractionation of the n-BuOH and EtOAc fractions was performed to obtain information about the nature of the chemical components of the plant extract, responsible for the antiviral effect. The individual constituents were identified by spectroscopic methods and comparison with authentic samples and by HPLC. The cell-toxic and virus-inhibitory effects of the fractions and some individual polyphenol compounds, found in Geranium sanguineum L., were studied using the replication of representative influenza viruses in cell cultures. This study showed that the presence of a variety of biologically active compounds as well as the possible synergistic interactions between them seem to be decisive for the overall antiviral effect.

This study was conducted to evaluate the radical scavenging capacities of extracts and preparations from a Lavandula vera MM plant cell culture with different rosmarinic acid content and to compare them with pure rosmarinic and caffeic acids as well. The methods, which were used are superoxide anion and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radicals scavenging assays. Results showed that extracts and preparations from Lavandula vera MM possess strong radical scavengers, as the best both radical scavengers appeared to be the fractions with enriched rosmarinic acid content, obtained after ethylacetate fractioning (47.7% inhibition of superoxide radicals and 14.2 μᴍ 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid equivalents, respectively). These data reveal the possibilities for application of these preparations as antioxidants.

The insecticidal properties of the crude extracts of the leaves and flowers of Anemone pavonina were evaluated on Pheidole pallidula ants and showed significant levels of activity. Bioassay-guided fractionations led to the isolation of the butenolide ranunculin (1) as the active principle. Chemical investigations of the extracts showed them to contain as major components the sitosterol glycopyranoside lipids 2-5 and the glycerides 6-8. The structures of the metabolites were elucidated, following acetylation and hydrolysis of the natural products, by interpretation of their NMR and mass spectral data. The uncommon lipid metabolites 2-8 were isolated for the first time from the genus Anemone and this is the first report of insecticidal activity of the Anemone metabolite ranunculin against ants.

Acetone extracts from eggplant (Solanum melongena L.) and violet pepper (Capsicum annuum L.) peels both belonging to the Solanaceae plant family were characterized with respect to their anthocyanin profiles, colour qualities and antioxidant capacities. According to HPLC-DAD-MS 3 analyses the major anthocyanin in eggplant was delphinidin-3-rutinoside, while the predominant pigment in violet pepper was assigned to delphinidin-3-transcoumaroylrutinoside- 5-glucoside. Since virtually all anthocyanins were delphinidin-based, the effect of acylation and glycosylation patterns on colour stability and antioxidant capacity could be assessed. Application of two in vitro-assays for antioxidant capacity assessment revealed that eggplant generally exhibited higher values compared to violet pepper which was ascribed to 3,5-diglycosylated structures predominating in the latter. The higher extent of acylation in violet pepper was reflected by a more purplish colour shade of the extracts, but did not translate into a higher stability against fading which again was attributed to additional glycosyl substitution at C5. These findings support the relevance of structurerelated activities of anthocyanins both for understanding food colour and their particular nutritional value.

The structure-activity relationship of 12 isolated acetylenes from galls of Hedera rhombea (Araliaceae) induced by Asphondylia sp. (Cecidomyiidae) and their derivatives has been studied for the inhibition of the shoot and root growth of rice, perennial ryegrass, cockscomb, lettuce, and cress. Almost all acetylenes generally showed growth inhibitory activity. The diacetylenes exhibited higher activity than the monoacetylenes, suggesting that a conjugated diyne segment is essential for the activity. On the other hand, the acetylenes with a nonoxidated methylene group at C-8 showed stronger activity comparing with those possessing hydroxy and acetoxy groups at C-8. Furthermore, it has been demonstrated that the acetylenes bearing a terminal olefinic group at C-16,C-17 enhanced the activity. It is thus clarified that important sites for the activity of the acetylenes from galls of H. rhombea are a conjugated diyne and a terminal olefinic group connecting to the aliphatic chain and that less oxidated compounds show more activity.

Detergents including fatty acid salts act as surface-active agents and thus possibly damage the plasma membrane structures of aquatic organisms. Therefore, when excess, the houseused and industrial outflows of such detergents into aquatic environments may have considerable impacts on the ecosystem. In this study, we propose the use of green paramecia (Paramecium bursaria) for assessing the acute toxicity of eight fatty acid salts (Na and K salts of oleate, palmitate, laurate and myristate) under various water conditions. The Paramecium in the stationary phase were used for a toxicity assay carried out on 12-well microplates and the median lethal concentration (LC 50 ) was determined for each fatty acid salt. In the low mineral culture medium prepared with ultra-pure water, the LC 50 for each fatty acid ranged from 5.8 to 144 ppm (w/v). The toxic levels of fatty acid salts differed in the following order: laurate, myristate ≥ oleate, palmitate. The toxic levels of oleate and palmitate salts were ca. 10-fold lower than those of laurate and myristate salts. When river water and local tap water instead of ultra-pure water were used for culturing, the toxic levels of all fatty acid salts were drastically lowered compared to the low mineral condition by 30- to 100-fold (198-660 ppm, w/v). Similar detoxification effect was observed when Ca or Mg was added to the low mineral culture media, indicating that the toxicity of fatty acid salts can be notably lowered as the mineral content increases. As we demonstrated that toxicities of fatty acid salts can be lowered in river water and tap water compared to the low mineral condition, some chemical substances behave differently in the different water conditions. Therefore, the use of natural waters reflecting the real environmental conditions in further collection of data on the ecotoxicity impacts of variety of chemicals is highly encouraged.

Redox activity was measured in vegetative and generative apical parts (5 mm of the stem) and youngest leaves of winter (cv. “Górczański”) and spring (cv. “Młochowski”) rape. Both genotypes were cultured under the same growth conditions (17/15 °C day/night, 16 h photoperiod), but winter rape was additionally vernalized (5/2 °C day/night, 56 days) in order to induce the generative development. The cyclic voltammetric method was used to measure the redox potential of samples in the presence of Fe 3+ ions. Changes in the redox activity were compared with changes in riboflavin content and activities of antioxidative enzymes: superoxide dismutase (SOD) and catalase (CAT). The higher level of Fe 3+ ions and riboflavin detected in generative apices and leaves of winter and spring varieties indicated that electrons (and their donors) were present at a lower level in these organs in comparison with the vegetative ones. On the contrary, SOD and CAT activity were lower in generative than in vegetative organs. This confirms changes in the redox balance and involvement of oxygen radicals in the generative development of rape plants. The similarity of the measured parameters between winter and spring varieties indicates that the observed changes are independent of the way of generative induction (vernalization). Riboflavin can serve as one of the electron carriers between other oxidation-reduction substances.

The newly isolated from industrial wastewater Pseudomonas fluorescens strain HW-6 produced glycolipid biosurfactants at high concentrations (1.4-2.0 g l -1 ) when grown on hexadecane as a sole carbon source. Biosurfactants decreased the surface tension of the air/ water interface by 35 mN m -1 and possessed a low critical micelle concentration value of 20 mg l -1 , which indicated high surface activity. They efficiently emulsified aromatic hydrocarbons, kerosene, n-paraffins and mineral oils. Biosurfactant production contributed to a significant increase in cell hydrophobicity correlated with an increased growth of the strain on hexadecane. The results suggested that the newly isolated strain of Ps. fluorescens and produced glycolipid biosurfactants with effective surface and emulsifying properties are very promising and could find application for bioremediation of hydrocarbon-polluted sites.

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3α-tigloyloxytropine and 3-tigloyloxy- 6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l -1 ) and indole-3-acetic acid (IAA, 0.5 mg l -1 ) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.

Callus cultures of Vaccinium corymbosum var. bluecrop were optimized for their isoprene derivatives production by supplementing Schenk-Hildebrandt (SH) medium with constant concentration of kinetin (2.32 μᴍ) and two different amounts of selected auxins. Every auxin, except for IBA, used in 10-time higher concentration (2,4D, NAA, IAA, NOA) stimulated biosynthesis of β-sitosterol and inhibited triterpene synthesis. Quantitative analysis of isoprene derivatives in callus biomass collected on the 25 th day of the experiment proved that the analyzed callus of Vaccinium corymbosum var. bluecrop synthesized the highest amount of isoprene derivatives after subculturing on SH medium modified with 22.6 μᴍ of 2,4D and 2.32 μᴍ of kinetin.

The underlying method for obtaining a β-carotene-rich carotenoid-protein preparation and exopolysaccharides is the associated cultivation of the carotenoid-synthesizing lactose-negative yeast strain Rhodotorula rubra GED8 with the yogurt starter culture (Lactobacillus bulgaricus 2-11 + Streptococcus thermophilus 15HA) in whey ultrafiltrate (45 g lactose/l) with a maximum carotenoid yield of 13.37 mg/l culture fluid on the 4.5 th day. The chemical composition of the carotenoid-protein preparation has been identified. The respective carotenoid and protein content is 497.4 μg/g dry cells and 50.3% per dry weight, respectively. An important characteristic of the carotenoid composition is the high percentage (51.1%) of β- carotene (a carotenoid pigment with the highest provitamin A activity) as compared to 12.9% and 33.7%, respectively, for the other two individual pigments - torulene and torularhodin. Exopolysaccharides (12.8 g/l) synthesized by the yeast and lactic acid cultures, identified as acid biopolymers containing 7.2% glucuronic acid, were isolated in the cell-free supernatant. Mannose, produced exclusively by the yeast, predominated in the neutral carbohydrate biopolymer component (76%). The mixed cultivation of R. rubra GED8 with the yogurt starter (L. bulgaricus 2-11 + S. thermophilus 15HA) in ultrafiltrate under conditions of intracellular production of maximum amount of carotenoids and exopolysaccharides synthesis enables combined utilization of the culture fluid from the fermentation process.

Glyphosate showed a remarkable effect inducing the change of flower symmetry from the actinomorphic to the zygomorphic type in Petunia hybrida. Glyphosate [N-(phosphonomethyl) glycine] reduced the anthocyanin content and showed a weak inhibitory effect against phenylalanine ammonia-lyase (PAL) activity. ʟ-2-Aminooxy-3-phenylpropionic acid (APA), an inhibitor of PAL activity, reduced the anthocyanin content but had no effect on flower shape. Additional phenylalanine or trans-cinnamic acid, the intermediates of glyphosate inhibition against PAL activity, could not recover the change of flower shape induced by glyphosate. These results suggested that the reduction of PAL activity alone could not account for the two characteristic changes of flower symmetry and pigmentation induced by glyphosate. On the other hand, the results of application of glyphosate-related compounds suggested that the structure of glyphosate contributed to induce the morphological change of Petunia flower. Glyphosate may thus be a very useful agent in the elucidation of unresolved questions of flower morphogenesis and the related metabolism.

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Insects of the genus Agathemera (Phasmatidae) live in the Chilean Andes over 1500 m above sea level. On disturbing, the insects release a spray that can cause temporary blindness in humans. Very little is known on the composition of chemicals used for defense by South American phasmids. Here we show that both female and male defensive secretion of Agathemera elegans is made up of 4-methyl-1-hepten-3-one. The compound is reported for the first time as a natural product.

β-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing β-ᴅ-glucose residues from the end of various oligosaccharides with the release of β-ᴅ-glucose. CelB gene, encoding the thermostable β-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express β-glucosidase in silkworm. The recombinant β-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed β-glucosidase was obtained at pH 5.0 and about 105 °C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant β-glucosidase were similar to the native counterpart. The expressed β-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable β-glucosidase using this system.

Chloral-derived β-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson’s disease. The cytotoxicity of 1-trichloromethyl- 1,2,3,4-tetrahydro-β-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED 50 value of 230 μᴍ. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-β-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the β-carboline ring ‘plane’ due to the high steric demand of the huge dichloromethyl group at C(1).

Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates - hydrazinium derivatives) was measured in vitro in a batch reactor at 25 °C, pH 8, ionic strength 0.11 ᴍ and enzyme activity 3.5 U by four nondependent analytical methods. Sevin®, rivastigmin (Exelon®) and galantamin (Reminyl®) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k 3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl- N′-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.