Band 64, Heft 1-2

Terpinen-4-ol is a monoterpenoid alcohol and component of the essential oils of several aromatic plants. Similarly to terpinen-4-ol, other monoterpenoid alcohols have shown anticonvulsant activity in convulsion animal models. The present study aimed to investigate the anticonvulsant activity of terpinen-4-ol. Treatment of mice with terpinen-4-ol ( 200 mg/kg) caused a signifi cant decrease in the spontaneous motor activity at 30, 60 and 120 min after administration. Terpinen-4-ol (100 and 200 mg/kg) produced a significant dosedependent increase in the duration of sleeping in mice. Pretreatment of mice with terpinen-4- ol at doses of 100, 200 and 300 mg/kg significantly increased the latency of pentylenetetrazole -induced convulsions. Terpinen-4-ol (200 and 300 mg/kg) also inhibited the induced seizures of picrotoxin. In another model, maximal electroshock seizure, terpinen-4-ol decreased the tonic hind convulsions percentage at the dose of 300 mg/kg. From the overall results we can conclude that terpinen-4-ol showed a depressant effect on the central nervous system and significant anticonvulsant activity.

A number of N-substituted thymine and adenine derivatives, 2a, b and 3a, b, were synthesized by the coupling reaction of 1-bromo-2,2-diethoxyethane with the corresponding base. The corresponding peptide nucleic acid (PNA) analogues, N-substituted ethylamino-3-hydroxypropanoate derivatives 5a, b and ethylamino-3-hydroxybutanoate derivatives 6a, b, were synthesized from the corresponding 2-[3,4-dihydro-5-methyl-2,4-dioxopyrimidin-1(2H)- yl]-acetaldehyde (3a) and 2-[6-amino-4H-purin-9(5H)-yl]-acetaldehyde (3b), respectively. The synthesized compounds were tested for their antiviral activity against hepatitis B virus (HBV). The plaque reduction infectivity assay was used to determine the virus count reduction as a result of the treatment with the tested compounds.

A new natural compound, named 6-O-(3’’,4’’-dimethoxycinnamoyl) catalpol, was isolated from the defatted alcoholic extract of the flowering parts of Buddleja asiatica Lour. (family Scrophulariaceae). Other separated known compounds included steroids (β-sitosterol, stigmasterol, stigmasterol-O-glucoside, β-sitosterol-O-glucoside), iridoid glucosides (methyl catalpol, catalpol, aucubin), phenylpropanoids (isoacteoside and acteoside), a triterpene saponin (mimengoside A), flavonoids (diosmin and linarin) in addition to the free sugars mannitol and sucrose. The structures of the isolated compounds were established by 1 H and 13 C NMR and mass spectrometry. Furthermore, the polar fraction of the flowering parts and the roots showed substantial antihepatotoxic activity comparable to that of the lignan silymarin.

The aerial parts of Satureja spicigera were collected at full flowering stage at Gazvin, Iran. The essential oil was isolated by hydrodistillation and analyzed by a combination of capillary GC and GC-MS. Fourteen compounds were identified, of which carvacrol (53.74%) and thymol (36.03%) were the main constituents, representing 99.12% of the total oil. The in vitro antibacterial activity of the essential oil was determined against six ATCC standard bacterial strains (Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) using disc diffusion as well as measurement of minimum inhibitory concentrations. The disc diffusion results and MIC values indicated high inhibitory activity against the test bacteria. The most susceptible organisms were the Gram-positive B. subtilis and S. aureus followed by E. faecalis, usually resistant to most common antibiotics. Among the Gram-negative bacteria, E. coli and K. pneumoniae were highly sensitive to the different oil concentrations in the disc diffusion method. Finally, P. aeruginosa, a highly resistant organism to most antibiotics, showed moderate susceptibility to Satureja spicigera essential oil.

Bioactivity-guided fractionation of extracts from the fungus IBWF79B-90A resulted in the isolation of three known naphthoquinones, herbarin, dehydroherbarin, and O-methylherbarin and the azaanthraquinone scorpinone as well as three structurally related derivatives, O-phenethylherbarin and herbaridines A and B. All seven compounds exhibited cytotoxic activities against several cell lines.

1 The purpose of the present study was to investigate antitumour and anti-inflammatory activities of flavonoids isolated from Byrsonima crassa, Davilla elliptica and Mouriri pusa. The antitumour activity was measured by the MTT assay in murine mammary tumour cells (LM2) and the IC 50 values of the fl avonoids tested ranged from (31.5 ± 2.97) to (203.1 ± 5.9) μg/ml. The fl avonoids (myricetin-3-O-α-L-rhamnopyranoside) and 3 (quercetin-3-Ogalactopyranoside) from D. elliptica were the most active ones against the tumour cells. The same samples were tested to determine the inhibition of the release of nitric oxide (NO) and of the tumour necrosis factor-alpha (TNF-α) in murine macrophages by the Griess and ELISA sandwich assay, respectively. Almost all the samples showed inhibitory activity to the release of NO but not of TNF-α. Of all substances tested, flavonoids 2 (quercetin) and 6 (myricetin) may show promising activity in the treatment of murine breast cancer by immunomodulatory and antiproliferative activities.

Two new mexicanolide-type limonoids, named xylomexicanin A (1) and xylomexicanin B (2), were isolated from seeds of the Chinese mangrove Xylocarpus granatum. Their structures were elucidated on the basis of spectroscopic methods. Compound 1 exhibited antiproliferative activity against human breast carcinoma cells (KT), while 2 did not show inhibitory effects on eleven human tumour cell lines tested.

1 Two 11(15→1)abeotaxanes having a tetrahydrofuran ring along the carbon atoms C-2, C-3, C-4, C-20 were identified for the first time from the needles of the Canadian yew, Taxus canadensis. The compounds could be identified as 4α,10β,13α-triacetoxy-15-benzoyloxy-2α,20β- epoxy-11(15→1)abeotax-11-ene-5α,7β,9α-triol () and 4α,7β,9α,10β,15-pentaacetoxy-2α,20β- epoxy-11(15→1)abeotax-11-ene-5α,13α-diol ( 2 ) on the basis of 1D-, 2D-NMR evidence and high-resolution FABMS analysis. Compound 1 showed weak growth inhibitory activities against T-98 and MM1-CB cells in vitro.

The occurrence of sesquiterpenes was investigated with the geosmin-producing cyanobacterium Calothrix PCC 7507. The essential oil obtained by vacuum destillation was studied in more detail by GC-MS methods and superposition with authentic compounds. Geosmin was the dominating compound while the other sesquiterpenes were minor components. Sesquiterpenes that have not been described before in cyanobacteria were isodihydroagarofuran, eremophilone and 6,11-epoxyisodaucane. Closed-loop stripping analysis revealed that most of the sesquiterpenes were found in the biomass of Calothrix, while eremophilone was mainly observed in the medium of the axenic culture. Eremophilone showed acute toxicity (LC 50 ) against Chironomus riparius (insecta) at 29 μ M and against Thamnocephalus platyurus (crustacea) at 22 μ M . The compound was not toxic for Plectus cirratus (nematoda). 6,11-Epoxyisodaucane and isodihydroagarofuran exhibited no toxicity to invertebrates when applied in concentrations up to 100 μ M .

1 n-Hexane and ethyl acetate extracts as well as coumarin derivatives obtained from the n-hexane extract of the aerial parts of Seseli gummiferum Pall. ex Sm. subsp. corymbosum (Boiss. & Heldr.) P.H. Davis (Apiaceae) were evaluated in vivo for their anti-inflammatory and antinociceptive activities. The n-hexane and ethyl acetate extracts of the species were shown to possess significant inhibitory activity against the carrageenan-induced hind paw edema and p-benzoquinone-induced writhing models in mice. Among the isolated coumarin derivatives; (−)-(3’S,4’S)-3’-acetoxy-4’-isovaleryloxy-3’,4’-dihydroseselin (), (−)-(3’S,4’S)-3’- acetoxy-4’-angeloyloxy-3’,4’-dihydroseselin ( 2 ), (+)-(3’S,4’S)-3’-hydroxy-4’-angeloyloxy-3’,4’- dihydroseselin (D-laserpitin) ( 3 ), (−)-(3’S,4’S)-3’-angeloyloxy-4’-hydroxy-3’,4’-dihydroseselin ( 4 ), and osthole ( 5 ), only the 3’-acetoxy derivatives 1 and 2 were found to possess potent antinociceptive and anti-infl ammatory activities, per os, without inducing any apparent acute toxicity as well as gastric damage, while all other compounds and extracts were found to be ineffective in the TPA-induced mouse ear edema model assay.

Synergistic interactions between antioxidants have been postulated but not proven. On the contrary, it has been reported that the antioxidant activity of mixtures of antioxidants can be lower than the sum of the antioxidant activities of individual components. We report that such a situation can be observed in 2,2ʹ-azobis(2-amidinopropane) dihydrochloride (AAPH)- treated phosphatidylcholine liposomes in which lipid peroxidation was monitored by oxidation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591). Glutathione, present inside liposomes, and hydrophobic antioxidants, present in the lipid bilayer, protected against lipid peroxidation, but their simultaneous action was lower than the sum of individual contributions. A possible explanation for this effect is proposed.

Cell growth and flavonoid production in cell suspension cultures of Glycyrrhiza inflata Batal were investigated under various initial inoculum densities, and sucrose and nitrogen concentrations to develop an optimization method for an improved flavonoid production. Both biomass accumulation and flavonoid production exhibited an “S” curve in one culture cycle, with the greatest value obtained on day 21, which showed that cell growth and fl avonoid biosynthesis went along isochronously. Moreover, according to the biomass and flavonoid production, the appreciate inoculum density, and the sucrose and nitrogen concentrations were 50 g FW L -1 , 50 g L -1 and 120 mmol L -1 , respectively. In addition, cell growth and flavonoid production showed a peak of 16.4 g DW L -1 and 95.7 mg L -1 on day 21 under the optimizing conditions, respectively. The flavonoid productivity of the cells which were cultured for 3 years is higher than that of the 3-year-old plant, which suggested that flavonoid production by cell cultures of G. inflata is a potentially profitable method. Therefore, this work is considered to be helpful for efficient large-scale bioprocessing of cell cultures in bioreactors.

Justicidin B, an arylnaphthalene lignan, has strong cytotoxicity on chronic myeloid and chronic lymphoid leukemia cell lines. The first report of the production of justicidin B in a Linum species concerned in vitro culture of Linum austriacum. Therefore, culture characterization and presence of arylnaphthalene-type lignans in calli and plantlets of Linum tenuifolium from section Linastrum, Linum bienne, and Linum glaucum from section Linum were studied. Seed germination of L. tenuifolium in the light and darkness was significantly higher (p < 0.05) than of L. bienne in the light and L. glaucum in the darkness. L. tenuifolium seedling length in the darkness was significantly higher (p < 0.01) than under light conditions. There were no significant differences in the calli and shoot biomass weight, number and length of shoots in three species over one month, while the shoot diameter of L. bienne was significantly different (p < 0.05) from that of L. glaucum. Justicidin B was detected in L. glaucum callus and plantlet cultures by HPLC/MS/UV-DAD and HPLC coupled with a photodiode array detector. This finding is important from pharmaceutical point of view and shows the chemosystematic relation between L. glaucum and L. austriacum and this method will be a powerful tool for detecting natural products in interested and endangered medicinal plants.

The changes in plant growth, transpiration rate, photosynthetic activity, plant pigments, electrolyte leakage, H 2 O 2 content, lipid peroxidation, catalase activity and endogenous content of abscisic acid (ABA) were followed in the leaves of two wheat varieties (sakha 93 and 94) during drought stress and subsequent rehydration. Drought stress caused several inhibitory changes in the growth of both wheat varieties, particularly in sakha 94. Exogenous ABA treatment improved the growth of sakha 93 plants as indicated by a higher relative water content, transpiration rate and lower electrolyte leakage and also enhanced the growth during the recovery period. Such improvement may be the result of the induction of enzymatic (catalase) and non-enzymatic (carotenoid) systems. ABA treatment did not ameliorate the negative effect of drought on the growth of sakha 94.

Analyses of the total pool of phenolic compounds and ferulic acid, as a photoprotector of the photosynthetic apparatus, and the activity of L-phenylalanine ammonia-lyase (PAL), as a key enzyme in phenolics synthesis, were carried out. Measurements were performed on drought-resistant (CHD 12, CHD 147) and -sensitive (CHD 220, CHD 247) genotypes of spring triticale during flowering under increasing leaf water deficit. Additionally, the emission of blue and red fluorescence from leaves were estimated. The exclusively in the resistant triticale genotype CHD 247 observed simultaneous increase in the content of ferulic acid and the total pool of phenolic compounds as a response to the leaf water deficit seems to be a promising biochemical indicator for a reliable selection of genotypes most resistant to drought stress. For the other genotypes, an increase in the total pool of phenolic compounds is accompanied by a decrease in the content of ferulic acid. An increase in the emission of red fluorescence, correlated with the high content of phenolic compounds, indicates the possibilities of these substances participating in the mechanisms of adaptation of the photosynthetic apparatus to water deficit in leaf tissues.

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m -1 with phenanthrene to 49.6 mN m -1 with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m -1 . The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a signifi cant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0 - 7.2 and temperature of (28 ± 2) °C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

The reaction between [PtCl 2 (DMSO) 2 ] and L-cysteine (L-Cys) has been investigated in the presence of micelles of sodium dodecyl sulfate (SDS) - as a model for biological membranes. Additionally, the inhibitory effect of [PtCl 2 (DMSO) 2 ] on the Na+,K+-ATPase activity and its partial prevention with 10 mM L-Cys were demonstrated. The interaction of L-Cys with [PtCl 2 (DMSO) 2 ] resulted in the formation of a [Pt(DMSO) 2 (L-Cys) 2 ] 2+ (DMSO) 2 ] complex, which most probably occurs through stepwise replacement of Cl- with L-Cys. It has also been demonstrated that neither the pH value nor SDS affects the composition of the new complex. On the other hand, the pH value and SDS do affect the reaction rate, most probably due to electrostatic interactions with reactants. In summary, this study can be used as a simple model approach for the investigation of reaction mechanisms between platinum complexes and various biomolecules, and for the determination of potential toxicity and/or side effects of antitumour platinum drugs.

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in the tumour cells of a patient, resulting from enhanced drug efflux. It is often related to the overexpression of P-glycoprotein (P-gp) on the surface of tumour cells, thereby reducing drug cytotoxicity. In the present study, naringin (the predominant flavonone found in grapefruit and other related citrus species) was tested for its potential ability to modulate the expression of P-gp in a short-term animal bioassay, in comparison with verapamil (a calcium channel blocker and positive MDR reversal agent). Western blot analysis showed that pre-treatment by i.p. administration of 5 mg naringin/kg body weight for 3 consecutive days prior to doxorubicin (the most common used anticancer drug which induces MDR) administration was able to significantly lower the P-gp expression reaching nearly the level of animals treated with verapamil. Moreover, pre-treatment with naringin prior to doxorubicin increased the sensitivity to the drug. Naringin inhibited the doxorubicin-stimulated ATPase activity demonstrating that naringin may interact directly with the transporter. In addition, the results demonstrated that induction of both glutathione (GSH) and glutathione-S-transferase (GST) by doxorubicin is consistent with an increased ATP-dependent doxorubicin transport. Thus, naringin seems to modulate the in vivo expression of P-gp. In summary, the present study describes the dual modulation of P-gp expression and function by the flavonoid naringin, which may be an attractive new agent for the chemosensitization of cancer cells.

Both light-dependent and light-independent (dark) protochlorophyllide (Pchlide) reductase account for catalyzing the reduction of Pchlide to chlorophyllide during the biosynthesis of Mg-tetrapyrrole pigments in cyanobacteria. To gain more insight into the interaction between the wavelength of the light and these two chlorophyll synthetic pathways in Synechocystis sp. PCC 6803, the spectral effectiveness of the formation of chlorophyll a was investigated during the regreening process in chlL - and chlN - mutants, which could not synthesize chlorophyll during growth in the dark. The action spectra showed obvious maxima around 450 nm and 650 nm, similar to those of higher plants except that the intensities of two peaks are reversed. The mRNA levels of chlL and chlN and chlorophyll a content under different wavelengths of light in the wild-type strain were also measured. The RT-PCR analysis revealed that the transcripts of chlL and chlN were up-regulated in red light but simultaneously down-regulated in green light which resulted in corresponding changes of the chlorophyll content. This fact indicates that the regulation of dark-operative protochlorophyllide oxidoreductase (DPOR) in the transcriptional level is essential for cyanobacteria to synthesize appropriate chlorophyll for acclimating in various light colour environments.

A simple, quick and efficient method for isolating total RNA from heavy browning cells was developed by adding polyvinylpyrrolidone, mercaptoethanol and 3 M NaAc during the process of the Trizol (a kind of a widely used RNA extraction buffer) method. High-quality total RNA was isolated and synthesized to cDNA. Transcript levels of four paclitaxel biosynthetic pathway genes: dxr, hmgr, ggpps and dbat were assayed by real-time RT-PCR. The results demonstrated that the transcript levels of these genes experienced a coincident descent in the past three years as well as a decreasing paclitaxel productivity. According to these results, the possible reason for the descending paclitaxel productivity during long-term Taxus media cv. Hicksii cell culture maybe due to a decreasing transcripts level of mass genes in close with a gross secondary metabolite level. Gene manipulation emphasized only on key enzyme genes in the paclitaxel biosynthesis pathway may not hamper the somaclonal variation trend of Taxus media cv. Hicksii cell culture.

A solid-state bioprocess with wheat bran and rice husk as substrate was used to isolate filamentous fungi with lipase activity from dairy effluent and soil contaminated with diesel oil. The lipase activity was measured in units, with one unit (U) being defined as the amount of enzyme required to liberate 1 μmol of fatty acids per minute per gram of bran substrate (1 U = 1 μmol min -1 g -1 ). We obtained 24 isolates of filamentous fungi with lipase activity, 17 from the dairy effluent and 7 from the diesel oil-contaminated soil. The best lipase producers were the dairy effluent isolate Aspergillus E-6, with a maximum lipase activity of 49.81 U, and Aspergillus isolate O-4 recovered from the diesel oil-contaminated soil, with a maximum lipase activity of 45.49 U. Both isolates produced their maximum lipase activity eight days after the start of the bioprocess.

A full-length cDNA (LC3ʹH) was obtained from a cDNA library of Lycoris radiata by DOP-PCR (degenerate oligonucleotide primer PCR), 3ʹrace and 5ʹrace methods. Compared with the other reported enzymes from different plants, the deduced amino acid sequence of LC3ʹH exhibits significant homologies to 3ʹ-hydroxylases that are involved in the caffeic acid biosynthesis. These findings suggest that the new gene is closely related to the biosynthesis of caffeic acid, which is also an important step of the galanthamine biosynthesis in Amaryllidaceae plants.

Three known nucleosides were isolated from the sponge Haliclona sp. The structures were established on the basis of NMR data and comparison with those reported, and chemotaxonomic relationships of the sponge nucleosides were discussed.

The amphiphilic polyzwitterion (PZ) poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl) ammonium propanesulfonate), zwitterionic surfactant (ZS) n-dodecyl- N,N-dimethyl-3-ammonium-1-propanesulfonate, and zwitterionic monomer (ZM) N,Ndimethyl( methacryloyloxyethyl)ammonium propanesulfonate were analyzed for their suggested chaperone-like effect on the interaction of C1q and IgG. Our results proved that the PZ retarded the C1q interaction with IgG, demonstrating a specific protein-folding helper effect. The ZS enhanced this interaction, when the ZS concentration was lower than the critical micelle concentration (CMC), and retarded it, when the ZS concentration was above the CMC. The ZM, with no self-assembling ability, did not influence this interaction. These results support the hypothesis of a hydrophobic interaction between Pts and hydrophobic domains of partly denatured protein molecules. The amphiphilic self-assemblies, formed by polyzwitterionic macromolecules or zwitterionic surfactants, have the ability to transform the hydrophobic domains of the protein molecules into hydrophilic ones, covering them with their hydrophilic parts.