Band 60, Heft 7-8

The resin of the tree Araucaria araucana (Araucariaceae) is used by the Mapuche Amerindians in southern Chile and Argentina to treat ulcers and has been shown to display a gastroprotective effect in animal models. A study was undertaken to isolate, identify and assess the gastroprotective effect of the resin constituents and its semisynthetic derivatives as well as to evaluate the cytotoxicity of the products in cell cultures. Eleven diterpenes (ten labdane and a pimarane) were isolated from a resin sample collected in Chile. The labdane derivatives 15-acetoxylabd-8(17)-en-19-ol as well as 15,19-diacetoxylabd-8(17)-en are reported for the first time as natural products. Six diterpenes previously described from other plant sources are reported for the first time for the A. araucana resin. The structure of all compounds was elucidated by spectroscopic means. Some 24 diterpenes isolated/prepared in amounts over 10 mg were evaluated for gastroprotective effects in the ethanol/HCl-induced ulcer model in mice at 100 mg/kg. The highest gastroprotective activities were provided by 15-hydroxyimbricatolal, 15-acetoxyimbricatolal, 15-acetoxylabd-8(17)-en-19-oic acid methyl ester and 15-acetoxy- 19-labdanoic acid, all of them being as active as the reference drug lansoprazole at 20 mg/kg. The cytotoxicity of 30 diterpenes as well as lansoprazole was assessed towards human lung fibroblasts (MRC-5) and 26 compounds were evaluated on the human gastric epithelial cell line AGS by means of the neutral red uptake assay. A concentration-dependent cell viability inhibition was found with IC 50 values ranging from 27 up to > 1000 μm. The relationship between the cytotoxicity data and lipophilicity of the products is also discussed.

A new prenylated depsidone, atrovirisidone B (2), together with naringenin (3) and 3,8″-binaringenin (4) were isolated from the roots of Garcinia atroviridis. Their structures were determined on the basis of spectral data interpretation. Compound 2 showed cytotoxic activity against human breast (MCF-7), human prostate (DU-145) and human lung (H-460) cancer cells.

Nematicidal bioassay-guided fractionation of the n-hexane extract of the seeds of Jubaea chilensis led to the identification of eight known fatty acids and a mixture of triglycerides, reported for the first time for this species. In addition, their corresponding methyl esters were identified to be artifacts generated during the extraction and isolation procedures by using GC-EI-MS and chemical transformation methods. The fatty acid composition of the triglycerides was analyzed by GC-EI-MS and chemical transformation techniques. Among the 17 compounds, only lauric acid and myristic acid exhibited significant inhibitory effects on the movement of Caenorhabditis elegans with minimum inhibitory concentrations (MIC) of 75 μg/ml.

The in vitro antibacterial and antioxidant activity of the essential oil and its two main components of Grammosciadium scabridum Boiss. (Apiaceae) growing wild in Iran, as well as the composition of its essential oil were studied. A total of 19 compounds representing 99.9% of the oil has been identified. γ-Terpinene (73.5%), p-cymene (14.2%) and (E)-β-farnesene (5.3%) were characterized as the main components. The oil showed remarkable activity against three Gram-negative and four Gram-positive test bacteria, with minimal inhibitory concentration (MIC) values ranging from 0.31 to 10.00 mg/ml. The oil and its two main components were also subjected to screening for their possible antioxidant activity by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The free radical scavenging capacity of the oil was determined with an IC 50 value of 6.6 mg/ml.

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5- trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol- 5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL 50 = 2.8 d 0.3 μg/ml) than piplartine (DL 50 = 32.3 d 3.4 μg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC 50 values in the range of 0.7 to 1.7 μg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.

Negligent industrial development has greatly contributed to environmental pollution through the contamination of water and soil by xenobiotic organic chemicals. Remedial strategies to deal with chemical pollution require reliable methods to identify and monitor contamination, as well as effective procedures to attenuate or to eliminate the pollutant. In the food chain, plants are ideally placed as early bio-indicators of environmental pollution as they experience and respond to environmental toxicants sooner than organisms at higher trophic levels. Furthermore, some plants are capable of detoxifying anthropogenic chemicals by metabolic transformation and could prove useful for the remediation of contaminated water and soil: so-called phytoremediation. So far research technologies aimed at developing plants for bio-indication/bio-monitoring and for remediation have largely relied on standardised chemical and biochemical procedures to evaluate phytotoxicity, metabolic fate and persistence of organic pollutants in plants. The next stage in the evolution of these plant-based technologies is the improvement and optimisation of any innate phytoremediation activities identified in selected plants. In general, uptake followed by metabolism and compartmentation is responsible for the detoxification of organic xenobiotics in plants. These are complex cellular systems that may be organised in well-defined pathways and are often controlled by large families of genes. In order to elucidate complex traits such as detoxification, an emerging idea is to make use of global approaches such as the new “omic” technologies to identify molecular changes in plant tissues exposed to specific organic xenobiotics. From expression profiles at the messenger RNA level, transcriptomics permit the identification of function-related gene clusters and at the protein level proteomics provide information on where, when and at what level specific proteins accumulate. We conclude that these global approaches may be a useful way of widening screening capability to identify appropriate molecular markers that can be used to improve detoxification activity.

Inhibitors of p-hydroxyphenylpyruvate dioxygenase (HPPD) are bleaching compounds impairing the formation of colored carotenoids. This activity makes them promising candidates for herbicides. Detailed studies on enzyme-inhibitor complexes or on the binding niche of the enzyme have still to be performed. Enzyme preparation from plants is time-consuming and the yield is poor. This paper describes in relevant detail the preparation of recombinant enzyme from Arabidopsis thaliana with good yield and high specific activity.

A two-stage method for in vitro propagation of six Genista species from shoot tips was developed. Multiple microshoot cultures were obtained by growing the shoot tip explants on Schenk and Hildebrandt medium supplemented with 9.84 μm 6-(γ,γ-dimethylallylamino)- purine and 0.99 μm thidiazuron. The best shoot elongation was achieved on Schenk and Hildebrandt medium containing 4.92 μm indole-3-butyric acid. The rooting of shoots brought best effects (100%) on Schenk and Hildebrandt medium with 2.68 μm 1-naphthaleneacetic acid. HPLC analysis indicated that six-month-old regenerated plants as well as the herb of intact plants produced a rich set of simple flavones (derivatives of luteolin and apigenin) and isoflavones (derivatives of genistein, daidzein, formononetin and biochanin A). Multiple microshoot cultures of all species produced no simple flavones at all. In vitro shoots accumulated selectively a rich group of phytoestrogens in the form of aglucones, glucosides and esters (derivatives of genistein and daidzein). Cultures obtained in vitro synthesized many times more isoflavones than the intact plants. In all shoots which were micropropagated the dominating compound was genistin (e.g. shoots of G. tinctoria D ca 3281.4 mg per 100 g dry weight). Possible influence of tissue differentiation on isoflavone content under in vitro and in vivo conditions is discussed.

Potential antioxidative activities of three series of newly synthesized N-oxides were studied. Individual components in each of the series differed in the lipophilicities and number of free radical scavenging groups. Various methods were used to determine their antioxidative efficiencies: Prevention of erythrocyte membrane lipid oxidation induced by UV irradiation and chromogen experiments in which antioxidative efficiencies of compounds were compared to that of the standard antioxidant Trolox (a water-soluble vitamin E analogue). Additionally, some hemolytic (pig erythrocytes) and differential scanning calorimetry (DSC) measurements were performed to determine a mechanism of the interaction between membranes and N-oxides. It was found that N-oxides, especially those of long alkyl chains (> C 12 H 25 ), readily interacted with both, erythrocyte and liposomal membranes. No marked differences were found in their protection of erythrocytes against oxidation. In most cases inhibition of oxidation changed between 15% and 25%. Still, it was far better than in chromogen experiments where suppression of free radicals reached 20% in the best case. It may be concluded that antioxidative capabilities of N-oxides are moderate. Studies on the interaction mechanism showed that incorporation of particular compounds into model membranes varied. Hemolysing activities of compounds increased with the elongation of the alkyl chain but differed for corresponding compounds of particular series indicating that lipophilicity of compounds is not the only factor determing their interaction with erythrocyte membranes. DSC experiments showed that N-oxides, upon incorporation into 1,2-dipalmitoyl-3-snphosphatidylcholine liposomes, shifted the subtransition (Tp) and the main transition (Tm). The shifts observed depended on the alkyl chain length. The effects differed for each series. It seems that in the case of long alkyl chain compounds the domain formation may take place. Generally, the decrease of Tm was greatest for the same compounds that exhibited the best hemolytic efficacy. The same conclusion concerns the decrease of cooperativity of the main transition and the observed changes suggest an increase in membrane fluidity. Both, erythrocyte and DSC experiments seem to indicate that compounds of particular series incorporate in a somewhat different way into membranes.

Application of indole-3-acetic acid (IAA) with a pollen growth inhibitor, aspterric acid (AA), results in the recovery of normal pollen development. In contrast, application of gibberellin (GA 3 ) with AA do not induce normal pollen growth. In addition, application of different concentrations of IAA with AA shortens the period of growth from bolting to first flowering as compared to that treated with AA alone. Furthermore, stem length and number of flower bud treated with IAA and AA were similar to those of control. These results suggest, that IAA may play an important role in reproductive growth of A. thaliana.

Biosurfactant activity and naphthalene degradation by a new strain identified as Bacillus cereus 28BN were studied. The strain grew well and produced effective biosurfactants in the presence of n-alkanes, naphthalene, crude oil and vegetable oils. The biosurfactants were detected by the surface tension lowering of the medium, thin layer chromatography and infrared spectra analysis. With (2%) naphthalene as the sole carbon source, high levels of rhamnolipids at a concentration of 2.3 g l -1 were determined in the stationary growth. After 20 d of incubation 72 ± 4% of the initial naphthalene was degraded. This is the first report for a Bacillus cereus rhamnolipid producing strain that utilized naphthalene under aerobic conditions. The strain looks promising for application in environmental technologies.

Production of tanshinones (tanshinone I and IIA) was determined in roots of Salvia przewalskii micropropagated plants. It was found that the total tanshinone content (tashinone I and tashinone IIA) was dependent on the age of the analyzed plants. The roots of 2-year-old in vitro regenerated plants at flowering stage produced highest tanshinone levels (3.8 mg/g dry weight of tanshinone I and 7.6 mg/g dry weight of tanshinone IIA).

The use of conventional and unconventional reaction methodology for the hydrolysis of the acetate group in zaluzanin D (1) resulted in hydration of the 11,13 exocyclic bond along with deacetylation. But the microorganism E. coli selectively cleaved the acetate group to yield zaluzanin C (4).

This paper shows the changes of the volatile compounds from four Astragalus species at three phenological stages: leaf development, flowering and fructification, which might be connected with the plant defense. After GC/MS analyses of Astragalus glycyphyllos L., A. hamosus L., A. cicer L. and A. spruneri Boiss., different groups of volatile compounds were found: hydrocarbons, alcohols, aldehydes and ketones, esters, terpenes, chlorinated compounds, etc. Identified volatiles were used for a cluster analysis in order to make chemotaxonomic conclusions for these evolutionary different species.

The interrelationship of five medicinally important species of Typhonium (Araceae) including T. venosum, which was previously placed under the genus Sauromatum, was inferred by analysis of random amplified polymorphic DNA (RAPD). DNA from pooled leaf samples was isolated and RAPD analysis was performed using 20 decamer oligonucleotide primers. Out of a total of 245 bands amplified, 12 were found to be monomorphic while 233 bands were polymorphic including 86 species-specific bands. The genetic similarities were analyzed from the dendrogram constructed by the pooled RAPD data using a similarity index. The dendrogram showed two distinct clades, one containing T. roxburghii, T. trilobatum and T. venosum and the other containing the remainder two species, i.e. T. diversifolium and T. flagelliforme. Both the clusters shared a common node approx. at 23.7% level of similarity. The maximum similarity of 31.2% was observed between T. venosum and T. trilobatum. In view of its close genetic similarity with other members of Typhonium, transfer of Sauromatum venosum to the genus Typhonium and merger of the two genera was supported.

Microsatellites, tandem repeats of short nucleotide (1-6 bp) sequences, are the DNA marker of choice because of their highly polymorphic, ubiquitous distribution within genome, ease of genotyping through polymerase chain reaction (PCR), selectively neutral, co-dominant and multi allelic nature. Six microsatellites, three polymorphic and three monomorphic, have been characterized for the first time in a bamboo species, Bambusa arudinacea belonging to the family Poaceae. The number of alleles per locus ranges form 2 to 13. Allelic diversity ranges from 0.041 to 0.870. Polymorphic information content (PIC) values for two loci were > 0.3, an indicator of polymorphic allele. Cross species amplification has been tested in other 18 bamboo species. Monomorphic simple sequence repeats (SSRs) have been found to be cross amplified in most of the tested species while polymorphic ones in only three to four species. The utility of the SSR loci in genetic diversity study of B. arundinacea and other cross amplified bamboo species have been discussed.

Identified germplasm is an important component for efficient and effective management of plant genetic resources. Traditionally, cultivars or species identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and analysis of genetic variation within 34 rose cultivars through random amplified polymorphic DNA (RAPD) markers. Analysis was made by using twenty five decamer primers. Out of twenty five, ten primers were selected and used for identification and analysis of genetic relationships among 34 rose cultivars. A total of 162 distinct DNA fragments ranging from 0.1 to 3.4 kb was amplified by using 10 selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 34 rose cultivars form 9 clusters. The first cluster consists of eight hybrid cultivars, three clusters having five cultivars each, one cluster having four cultivars, two clusters having three cultivars each and two clusters having one cultivar each. The genetic distance was very close within the cultivars. Thus, these RAPD markers have the potential for identification of clusters and characterization of genetic variation within the cultivars. This is also helpful in rose breeding programs and provides a major input into conservation biology.

The variability level of the ISSR (inter-simple sequences repeat) primer (GACA) 4 was examined in the three Lepidoptera families Pyralidae, Sphingidae and Pieridae. Our study shows that the tetra-repeat (GACA) n is evidently present in sufficient numbers in these butterflies to provide informative DNA fingerprints. The variability is mostly rather high, but within a comparable range to other ISSR studies. Although less polymorphisms may be encountered in some butterfly families, this study indicates that high variability of this marker may be a common characteristic of Lepidoptera genomes. An appeal for a minimal level of standardization of ISSR-PCR data analysis is formulated to enable an exact comparison between the groups of organisms studied with this fingerprint technique.

Sex attractants were established for one Brachodidae, three Choreutidae and two Tineidae moth species during field screening tests with (2E,13Z)-octadecadien-1-al, (2E,13Z)-, (3E,13Z)-, (3Z,13Z)-octadecadien-1-ols and their acetates (2E,13Z-18:Ald, 2E,13Z-, 3E,13Z-, 3Z,13Z-18:OH/OAc) as well as of binary mixtures of these compounds in West-Kazakhstan and Lithuania. Males of Brachodes appendiculata were attracted by 3E,13Z-18:OAc, Prochoreutis ultimana and P. myllerana by 2E,13Z-18:OH, Monopis palidella by 2E,13Z-18:Ald and Triaxomera fulvimitrella by binary mixtures of 3Z,13Z-18:OAc with either 3E,13Z-18:OH in the ratio of 5:5 or 3Z,13Z-18:OH in the ratio of 9:1 (v/v). The 3-component mixture composed of 2E,13Z-18:OH, 3Z,13Z-18:OH and 2E,13Z-18:Ald in the ratio 1:1:1 was developed to attract Prochoreutis sehestediana males. Attraction antagonists for B. appendiculata, P. ultimana and M. palidella were shown.

In spite of the importance of chemoreception and chemical signals in the social organization of lizards, there are only a few studies examining the chemical composition of secretions of lizards used for scent marking. The secretion of the femoral glands of male Iberian rock lizards (Lacerta monticola cyreni) contains 44 lipophilic compounds, including several steroids (mainly cholesterol), and n-C 6 to n-C 22 carboxylic acids, and minor components such as esters of carboxylic acids, alcohols, squalene, and one lactone. These compounds were identified on the basis of mass spectra, obtained by GC-MS. Most lipids were detected in all individuals, although relative proportions of each chemical show a high interindividual variability. This variability might be related to the characteristics or physical and health condition of males and might be the basis of female choice based on chemical cues observed in this lizard species.

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 × 10 -8 for the 10 microsatellite set, 2.3 × 10 -11 for the 14 microsatellite set, and 1.4 × 10 -13 for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 × 10 -11 . The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.

A simple method for spontaneous transfection into mammalian cells (both adherent and suspension in culture) with plasmid DNA is described. This method does not require any specific DNA carrier or technical device and can be applied for obtaining both transient and stably transfected cells. The efficiency of spontaneous transfection is slightly lower in comparison with that of the conventional calcium phosphate and lipofectin transfection methods and does not depend on the type of cell culture used.

The interaction of phytoestrogens with the most important binding sites of steroid hormones, i.e. sex hormone-binding globulin and estrogen receptors, was investigated. Relative binding affinities and association constants for 21 compounds among them isoflavones, flavones, flavonols, flavanones, chalcones and lignans were determined. The lignan nordihydroguaiaretic acid weakly displaced 17β-[ 3 H]-estradiol from estrogen receptor and Scatchard analysis suggests non-conformational changes. Compounds from Glycyrrhiza glabra, liquiritigenin and isoliquiritigenin, showed estrogenic affinities to both receptors. 18β-Glycyrrhetinic acid displaced 17β-[ 3 H]-estradiol from sex hormone-binding globulin but not from the estrogen receptor. Phytoestrogens compete with 17β-estradiol much stronger than with 5α-dihydrotestosterone for binding to sex hormone-binding globulin.