Band 62, Heft 11-12

The complex pigment pattern of fly agaric (Amanita muscaria) cap skins has been studied by LC-DAD and mass spectrometry. Among the betaxanthins the corresponding derivatives of serine, threonine, ethanolamine, alanine, Dopa, phenylalanine and tryptophan are reported for the first time to contribute to the pigment pattern of fly agarics. Betalamic acid, the chromophoric precursor of betaxanthins and betacyanins, muscaflavin and seco-dopas were also detected. Furthermore, the red-purple muscapurpurin and the red muscarubrin were tentatively assigned while further six betacyanin-like components could not be structurally allocated. Stability studies indicated a high susceptibility of pigment extracts to degradation which led to rapid colour loss thus rendering a complete characterization of betacyaninlike compounds impossible at present. Taking into account these difficulties the presented results may be a starting point for a comprehensive characterization of the pigment composition of fly agarics.

Two new xanthones, characterized as 4-(1,1-dimethylprop-2-enyl)-1,3,5,8-tetrahydroxyxanthone (1) and penangianaxanthone (2), with three known xanthones, cudratricusxanthone H (3), macluraxanthone C (4) and gerontoxanthone C (5), as well as friedelin and stigmasterol were isolated from the leaves of Garcinia penangiana. Their structures were elucidated by analysis of spectroscopic data and comparison of the NMR data with the literature ones. Significant cytotoxicity against DU-145, MCF-7 and NCI-H460 cancer cell lines was demonstrated by compounds 1D5, with IC 50 values ranging from 3.5 to 72.8 μm.

Trypanocidal activity was found in the volatile oil of dried Dracocephalum kotschyi. GCMS analysis determined that the major constituents of the oil were geranial (35.8%), C 10 H 14 O (26.6%), limonene (15.8%) and 1,1-dimethoxy decane (14.5%). In order to isolate the unknown biologically active monoterpene, fractionation of the volatile oil was carried out by silica gel column chromatography. The structure of the oxygenated compound was confirmed to be limonene-10-al (C 10 H 14 O) by analysis of physical and spectroscopic data ( 1 H NMR, 13 C NMR, HMBC and HMQC).

Nine phenolic compounds, such as cis-/trans-p-coumaric acid, cis-/trans-p-coumaric acid methyl ester, glucose ester of cis-/trans-p-coumaric acid, caffeic acid methyl ester, kaempferol 7-O-β-d-glucoside and kaempferol 3-O-β-d-glucoside, were isolated from Lavatera trimestris flowers by chromatographic techniques and their structures were elucidated by spectral means (NMR). All compounds were tested for their antioxidant activity, while the methanolic extract was tested also for its antimicrobial activity. Also several non-polar constituents have been identified using GC and GC/MS methods. This is the first time that phenolic esters and non-polar constituents were identified in the flowers of L. trimestris L.

The ethanolic extracts of two Brazilian propolis samples were submitted to a fractionation procedure based on the pK a values of their components. The fractions obtained were evaluated for their antimicrobial activity against Staphylococcus aureus as well as for their antioxidant properties (reduction of DPPH radical). Their phenolic and flavonoid contents were measured spectrophotometrically, in order to establish the correlations between these contents and the measured activities. Further, the most active fractions of both extracts were analyzed by HRGC-MS and about twenty compounds could be characterized. Among them were 3-prenyl-4-hydroxycinnamic acid (drupanin) and 3,5-diprenyl-4-hydroxycinnamic acid (artepillin C), which seem to be the major antioxidant components of the bioactive fractions.

In the course of our search for new bioactive compounds from basidiomycetes, four new compounds were isolated from fermentations of Limacella illinita. Illinitone A (1) exhibited weak phytotoxic and moderate nematicidal activities against Caenorhabditis elegans, illinitone B (2) was moderately cytotoxic, while limacellone (3) exhibited weak cytotoxic and phytotoxic activities. The muurolane sesquiterpene 4a was found to be inactive in the assays performed here. Limacellone (3), which appeared to be related with the illinitones 1 and 2, has a new C 15 carbon skeleton. It is possible that compounds 1, 2 and 3 are terpenoids/ secoterpenoids, but their biosyntheses were not investigated.

The anti-inflammatory and antinociceptive properties of four major compounds from the flowers of Verbascum pterocalycinum var. mutense were investigated. Saponin glycosides called ilwensisaponin A and C and iridoid glycosides known as ajugol and picroside IV were isolated from the methanolic extract. A dose-related anti-inflammatory and antinociceptive response were obtained in this study at doses of 100 and 200 mg/kg. The results of the evaluation of the anti-inflammatory activity induced by carrageenan and PGE1 showed that this species possesses active constituents that could diminish the cyclooxygenase activitiy. No effects were observed in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model. Our results support the anti-inflammatory and analgesic effects of Verbascum pterocalycinum var. mutense. Ilwensisaponins A and C could explain in part the anti-inflammatory and analgesic activities of this species. Although antinociceptive and anti-inflammatory activities of ajugol and picroside IV were found insignificant in the statistical analysis, ilwensisaponin A and C showed notable activity without inducing any apparent acute toxicity as well as gastric damage.

By using a new bioassay-guided method, 2-hydroxy-4-methoxybenzaldehyde isolated from the root bark of Periploca sepium, a traditional Chinese medicine, showed repellent activity against the olive weevil (Dyscerus perforatus) at 62.5, 125, 250 and 500 μg/disc, respectively. In addition, it also exhibited antinematodal activity against Bursaphelenchus xylophilus at a minimum effective dose of 200 μg/ball. The three related compounds obtained were also evaluated for the above-mentioned bioactivities.

The study focussed on four limonoids (calodendrolide, harrisonin, pedonin and pyroangolensolide) as larvicidal components against mosquito 2 nd instar larvae of the species Aedes aegypti Linn. senso stricto. Since pyroangolensolide is close to calodendrolide in structure, it was synthesized through reduction of calodendrolide with chromium(II) chloride in acetone. Harrisonin and pedonin were extracted with cold methanol from the root bark of Harrisonia abyssinica while calodendrolide was extracted with the same solvent from the root bark of Calodendrum capense. The structure of pyroangolensolide was elucidated using physical and spectroscopic techniques. 25, 50, 75, and 100 μm of each compound were tested against the mosquito larvae. Calodendrolide was the most toxic since 100% mortality was registered at all concentrations, while pyroangolensolide showed 100% mortality up to 50 μm and for contents of 25 μm, a mortality of 70% was registered. As a result of this toxicity, lower concentrations (5, 10 and 15 μm) were tested for both calodendrolide and pyroangolensolide. Toxicity of harrisonin and pedonin was lower. The relative toxicity was in the order: calodendrolide > pyroangolensolide >harrisonin > pedonin with LC 50 values of 13.2, 16.6, 28.1 and 59.2 μm, respectively.

Alzheimer’s disease (AD), the most common form of dementia, is a neurodegenerative disease characterized by progressive cognitive deterioration together with declining activities of daily living and neuropsychiatric symptoms or behavioural changes. The oldest, on which most currently available drug therapies are based, is known as the “cholinergic hypothesis” and suggests that AD begins as a deficiency in the production of the neurotransmitter acetylcholine. Therefore, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors have gained a great popularity for the treatment of AD. In this study, we screened in vitro inhibitory activities of a number of phenolic acids (chlorogenic, caffeic, gallic, and quinic acids) as well as of various flavonoid derivatives (genistein, biochanin A, naringin, apigenin, quercetin, luteolin-7-O-rutinoside, kaempferol-3-O-galactoside, diosmin, silibinin, and silymarin) against AChE and BChE at 1 mg/ml concentration using a microplate-reader assay based on the Ellman method. Among them, only quercetin showed a substantial inhibition (76.2%) against AChE, while genistein (65.7%), luteolin-7-O-rutinoside (54.9%), and silibinin (51.4%) exerted a moderate inhibition on BChE.

This paper demonstrates for the first time that plant metabolites of the phenylamide type, conjugates of putrescine with hydroxycinnamic acids (p-coumaric, caffeic and ferulic), possess 1 O 2 quenching properties. Data were obtained confirming that their acidic parent compounds were also able to quench 1 O 2 , as did polyamines (putrescine, spermidine and spermine), and that this ability depends on the number of amino groups. Potentiation of the 1 O 2 quenching ability of the conjugates relative to both parent components was established. The importance of polyamines and phenylamides in the plant non-enzymatic antioxidant defence at sites of intensive 1 O 2 generation, such as the photosynthetic centers, was suggested.

The procedure of Salvia przewalskii shoot multiplication and the ability of regenerated plants to produce essential oil is reported. The essential oil was obtained by hydrodistillation from leaves and flowering stems of field-grown plants, and their chemical composition was examined by GC, GC-MS and 1 H NMR. The differences in yield as well as qualitative and quantitative composition between the oils isolated from in vitro and in vivo plants were observed. S. przewalskii essential oil was tested for its antimicrobial and cytotoxic properties. It was found that cytotoxicity against human leukemia HL-60 cells and antimicrobial activity (especially, against Staphylococcus aureus and S. epidermidis strains) of oils isolated from in vitro plants were higher than those for oils from in vivo S. przewalskii plants.

A change of flower shape was observed in petunia corollas treated with 0.5 mm glyphosate. Glyphosate changed the flower symmetry from the actinomorphic type to the zygomorphic type. Corollas treated with glyphosate showed an increased free amino acid content. Free amino acid profiles in petunia corollas revealed that glyphosate had no significant effect on aromatic amino acid levels but increased the level of proline. Soluble protein content in glyphosate-treated corollas did not cause any significant changes. The contents of soluble phenolics, lignin, and IAA in the corollas were not significantly affected by the glyphosate treatment. In contrast, glyphosate reduced the nitrate content and the RNA content of petunia corollas by 45% and 63% of the control, respectively. However, the DNA content in glyphosate-treated corollas was similar to that of the control. Low concentrations of glyphosate did not show any phytotoxic effects on the whole plants and any remarkable changes on aromatic amino acid metabolism and protein synthesis. However, glyphosate reduced the RNA content of petunia corollas and changed the flower symmetry from the actinomorphic type to the zygomorphic type. The results of nonprotein nitrogen metabolism in glyphosate-treated petunia corollas suggested that glyphosate application at low concentration may influence the regulation of flower symmetry through the change of RNA biosynthesis.

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.

The hemolymph chymotrypsin inhibitor b1 (CIb1) of silkworm, Bombyx mori, plays an important role in innate immunity. In order to study its encoding gene CIb1, five heterogeneous promoter fragments of 844 bp, 682 bp, 516 bp, 312 bp and 82 bp in length were cloned from genomic DNA of the p50 silkworm strain. Characterization of the CIb1 promoter was performed in vitro using the firefly luciferase gene as reporter. The results showed that CIb1 promoter fragments have transcription activities in the B. mori ovary-derived BmN cell line. The 82 bp fragment (-72 to +10 nt) containing the eukaryotic core promoter elements revealed a basic transcription activity. The Bm1 element, upstream the transcription initiation site, showed a positive regulation function to the CIb1 promoter. CIb1 promoter-like fragments from the genomic DNA of the tetra hybrid silkworm Suju\Minghu provided a natural deletion model for the study of the CIb1 promoter. In vitro analysis indicated that the 132 bp fragment from -517 nt to -386 nt upstream of the transcription initiation site strongly suppressed the transcription activity of the CIb1 promoter, suggesting that the 132 bp fragment harbours strong negative cis-acting elements. Infection of Bombyx mori nucleopolyhedrovirus (BmNPV) increased the activity of the CIb1 promoter, having provided another evidence to the function of CIb1 in the innate immunity of silkworm.

MSAR (1-sulfate-3-myristoyl-5-pentadecylbenzene) is a semisynthetic derivative of 5-npentadecylresorcinol (C15:0). MSAR exhibits hemolytic activity against sheep erythrocytes with a EH 50 value of (35 ± 1.7) μm. At low concentrations MSAR also exhibits the ability to protect cells against their hypoosmotic lysis. This protective effect is significant as, at 0.1 μm of MSAR, the extent of osmotically induced cell lysis is reduced by approx. 20%. It was demonstrated that the 9-anthroyloxystearic acid signal was most intensively quenched by MSAR molecules, suggesting a relatively deep location of these molecules within the lipid bilayer. MSAR causes an increase of the fluorescence of the membrane potential sensitive probe. This indicates an alteration of the surface charge and a decrease of the local pH value at the membrane surface. At low bilayer content (1-4 mol%) this compound causes a significant increase of the phospholipid bilayer fluidity (both under and above the main phase transition temperature) of dipalmitoylphosphatidylcholine (DPPC) liposomes. At this low content MSAR slightly decreases the main phase transition temperature (T c ) value. The effects induced in the phospholipid bilayer by higher contents of MSAR molecules (5-10 mol%) make it impossible to determine the Tc value and to evaluate changes of the membrane fluidity by using pyrene-labeled lipid. MSAR also causes a decrease of the activity of membrane-bound enzymes-red blood cell acetylcholinesterase (AChE) and phospholipase A 2 (PLA 2 ). MSAR decreases the AChE activity by 40% at 100 μm. The presence of MSAR in the liposomal membrane induces a complete abolishment of the lag time of the PLA 2 activity, indicating that these molecules induce the formation of packing defects in the bilayer which may result from imperfect mixing of phospholipids.

Peripheral blood cultures were exposed to various doses (5 to 500 mg/L) of boron compounds. Sister-chromatid exchange, micronucleus and chromosomal aberration tests were applied to estimate the DNA damage, and biochemical parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, total glutathione, malondialdehyde and total antioxidant capacity) were examined to determine oxidative stress. According to our findings, various boron compounds at low doses were useful in supporting antioxidant enzyme activities in human blood cultures. It was found that the boron compounds do not have genotoxic effects even in the highest concentrations, though in increasing doses they constitute oxidative stress. It is concluded that the tested boron compounds can be used safely, but it is necessary to consider the tissue damages which are likely to appear depending on the oxidative stress.

We studied the effect of some modulators of signal transduction on the erythrocyte Na + / K + -ATPase. Go6976 and Go6983 (protein kinase C inhibitors) showed a stimulatory effect and calyculin A (protein phosphatase inhibitor) exerted an inhibitory effect on the Na pump activity. Some of the tested modulators of cell-signaling [protein phosphatase(s), phosphodiesterase, calmodulin and some protein kinases] interfered with the lactoferrin (Lf) stimulatory effect on the sodium pump. Lf itself was able to modulate the effect of some agents upon the pump activity. Moreover, an additive effect of stimulation was found when Lf and some agents were used simultaneously. The summarized results showed that: (i) Lf upregulates the Na + /K + -ATPase in erythrocytes and facilitates the K + influx into the erythrocytes; (ii) the effect of pump stimulation is mediated by phosphorylation processes. These results suggest a potential opportunity for using Lf alone or together with other agents as a stimulator of the erythrocyte Na + /K + -ATPase.

Cisplatin [cis-diamminedichloroplatinum(II) or cis- DDP], but not its stereoisomer transplatin, is suggested to be among the most powerful anticancer agents. It is believed that its therapeutic activity results from its interaction with DNA forming intra- and interstrand crosslinks. During our earlier investigations, we have observed a prominent preference of the linker histone H1 for binding to cis-platinated DNA (containing several different cross-links along the DNA fragment) compared with unmodified or transplatin-modified DNA. This report presents our recent experimental data obtained by band-shift analysis on the binding of H1 to a cisplatin-modified synthetic 34 bp DNA fragment containing a single target d(GG/CC) for 1,2 cis-intra-platination. Results obtained with another nuclear protein with similar DNA-binding properties, HMGB1, are also presented. The experimental data throw light on the precise preference of histone H1 for binding to different types of cisplatin-created cross-links in DNA.

Eburicoic acid was produced and isolated from the cultured mycelia of the fungus, Trametes versatillis. The weight rate of eburicoic acid in the dry weight of cultured mycelia of Trametes versatillis was about to 1%. Trametes versatillis can be expected as an important bioresource to supply eburicoic acid for its industrial exploitation.