Band 63, Heft 7-8

In a bioassay-guided approach the chemical composition of rhizomes of Metaxya rostrata (Kunth C. Presl) was studied for the first time. Investigations of the cytotoxicity of extracts and fractions on SW480 colorectal carcinoma cells resulted in the isolation of two polyphenols D cinnamtannin B-1 and aesculitannin B. The structures of the compounds were elucidated by different NMR experiments. Additionally, sugars, common sterols, such as sitosterol, stigmasterol and campesterol, as well as chlorogenic acid and caffeic acid were identified in Metaxya rostrata.

The antioxidant activity of ethanol extracts of Atrichum undulatum, Polytrichum formosum (Polytrichaceae), Pleurozium schreberi (Entodontaceae) and Thuidium tamariscinum (Thuidiaceae) was evaluated by an electrochemical method (cyclic voltammetry) and standard photometric methods: Fe(III) to Fe(II) reducing power, nitric oxide scavenging (NO) assay and simulation of Fenton-type reaction by nonsite-specific (NSSOH) and site-specific (SSOH) hydroxyl radical-mediated 2-deoxy-d-ribose degradation inhibition. The total content of phenols was determined by the Folin-Ciocalteau reagent. All tested species showed antioxidant effects lower than the positive control, caffeic acid. The extracts of A. undulatum and P. formosum contained the highest content of phenols and were the most effective in Fe(III) to Fe(II) reducing power, cyclic voltammetry and SSOH assay. By contrast, only the extract of Pl. schreberi showed activity in the NSSOH assay. A. undulatum and T. tamariscinum extracts were the most active in the NO assay. The results suggest that the extracts of A. undulatum and P. formosum possess stronger antioxidant activity than those of Pl. scheberi and T. tamariscinum, but they affect the Fenton-type reaction mainly by iron chelation.

The methanol extract of the leaves of Buddleja asiatica Lour. (Loganiaceae) showed antioxidant activity toward the well known in vitro antioxidant tests such as total antioxidant capacity by the phosphomolybdenum method, free radical scavenging activity by the 1,1- diphenyl-2-picrylhydrazyl scavenging assay (DPPH assay) and hydrogen peroxide scavenging methods. Due to the high scavenging activity of the n-butanol successive fraction toward DPPH and H 2 O 2 (SC 50 = 11.99 and 18.54 μg/ml, respectively), this extract was subjected to chromatographic separation and isolation. Four non-phenolic compounds were isolated and identified on the basis of spectroscopic and chemical analyses: 1-O-ß-D-glucopyranosyl- 2-methoxy-3-(2-hydroxy-triaconta-3,12-dienoate)-glycerol (1), 3-O-[α-l-rhamnopyranosyl- (1→4)-ß-d-glucopyranosyl-(1→3)]-[ß-d-glucopyranosyl-(1→2)]-ß-d-fucopyranosyl-olean- 11,13(18)-diene-3ß,23,28-triol (2), 3-O-[α-l-rhamnopyranosyl-(1→4)-ß-d-glucopyranosyl- (1→4)-ß-d-glucopyranosyl-(1→3)]-ß-d-fucopyranosyl-olean-11,13(18)-diene-3ß,23,28-triol (3), and 3-O-[α-l-rhamnopyranosyl-(1→4)-ß-d-glucopyranosyl-(1→3)]-[ß-d-xylopyranosyl- (1→2)]-ß-d-glucuronopyranosyl-acid-olean-11,13(18)-diene-3ß,23,28-triol (4). The four compounds were evaluated as antioxidant agents using the three antioxidant bioassay tests.

Dunalia spinosa, a plant used in folk medicine for toothaches, breathing problems and cleansing wounds, was found active as antimicrobial and antioxidant. A new (E)-aurone rutinoside (dunaurone) has been isolated from the aerial parts of the plant, and its structure was determined by spectroscopic means. Lupeol, ß-sitosterol, scopoletin, quercetin and withaferin A were also found. All the extracts exhibited strong antimicrobial activity while dunaurone showed only weak antimicrobial inhibition against Klebsiella pneumoniae; in addition it presented a significant free radical scavenging activity.

Austroplenckia populnea (Celastraceae), known as “marmelinho do campo”, is used in Brazilian folk medicine as antimicrobial, anti-inflammatory, and antitumoural agent. The aim of the present work was to evaluate the antimicrobial, antileishmanial and antimalarial activities of the crude hydroalcoholic extract of A. populnea (CHE) and some of its isolated compounds. The phytochemical study of the CHE was carried out affording the isolation of methyl populnoate (1), populnoic acid (2), and stigmast-5-en-3-O-β-(d-glucopyranoside) (3). This is the first time that the presence of compound 3 in A. populnea is reported. The results showed that the CHE presents antifungal and antibacterial activities, especially against Candida glabrata and Candida albicans, for which the CHE showed IC 50 values of 0.7 μg mL -1 and 5.5 μg mL -1 , respectively, while amphotericin B showed an IC 50 value of 0.1 μg mL -1 against both microorganisms. Compounds 1D3 were inactive against all tested microorganisms. In the antileishmanial activity test against Leishmania donovani, the CHE showed an IC 50 value of 52 μg mL -1 , while compounds 2 and 3 displayed an IC 50 value of 18 μg mL -1 . In the antimalarial assay against Plasmodium falciparum (D6 and W2 clones), it was observed that all evaluated samples were inactive. In order to compare the effect on the parasites with the toxicity to mammalian cells, the cytotoxicity activity of the isolated compounds was evaluated against Vero cells, showing that all evaluated samples exhibited no cytotoxicity at the maximum dose tested.

Analysis by gas chromatography-mass spectrometry led to the identification of 26 compounds in Acorus gramineus essential oil. The antifungal activity of the identified compounds was tested singularly by using standard compounds. Allyl isothiocyanate and cis-asarone showed inhibition rates of 100% against P. cactorum at 28 mg/l air. In a test with C. parasitica and F. circinatum, allyl isothiocyante and cis-asarone showed moderate activity at 28 mg/l air.

Triterpenoids with 31-norcycloartanone structure were isolated for the first time from the Solanum genus. Cycloeucalenone and 24-oxo-31-norcycloartanone were the main constituents of the dichloromethane extract of Solanum cernuum Vell. leaves [7% (w/w) and 1.47% (w/w)]. Both triterpenoids were tested against human tumour cell lines, and 24-oxo-31-norcycloartanone was significantly active and selective against the lung tumour cell line NCI-H460 with total growth inhibition at 1.10 μg/mL, growth inhibition 50 at 0.19 μg/mL and lethal concentration 50 at 8.43 μg/mL, while cycloeucalenone showed poor activity. A homologous series of alkanes (C 25 -C 34 ), β-sitosterol, and the xanthophyll lutein were also identified. The antiulcer activity was assayed for the dichloromethane extract. In the gastric ulcer model induced by 95% ethanol, administration of 500, 1000 and 2000 mg/kg/po dichloromethane extract gave ulcer lesion indices of, respectively, 38.2, 61.0 and 81.9%, while carbenoxolone inhibited 88.9% at 200 mg/kg. In the gastric ulcer model induced by indomethacin the dichloromethane extract showed a small percentage of lesion inhibition. The ethanol extract was also analyzed and was mainly composed of glycoalkaloids, peptides and disaccharides.

Assay-guided fractionation led to the isolation of nine β-dihydroagarofuran sesquiterpenoids from the fruits of Celastrus orbiculatus. All isolated β-dihydroagarofuran sesquiterpenoids were tested for their cytotoxic activity against human melanoma A375-S2 and human cervical carcinoma Hela cell lines. Among them, compounds 1-5 and 7 showed cytotoxic activity. Compound 3 exhibited promising cytotoxicity against both human melanoma A375- S2 and human cervical carcinoma Hela cell lines. The structure-activity relationship was discussed briefly.

Nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-Nmethyl- nitramine (tetryl) comprise an important group of toxic environmental pollutants, whose toxicity is mainly attributed to the flavoenzyme electrontransferase-catalyzed redox cycling of their free radicals (oxidative stress) and DT-diaphorase [NAD(P)H:quinone oxidoreductase, NQO1, EC 1. 6.99.2]-catalyzed formation of alkylating nitroso and/or hydroxylamine metabolites. Because of the incomprehensive data on the immunotoxic effects of nitroaromatic explosives, we have studied the structure-cytotoxicity relationships in the action of tetryl, TNT as well as its amino and hydroxylamino metabolites, and related nitroaromatic compounds towards mouse splenocyte cells. The protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine against the cytotoxicity of TNT and other nitroaromatics showed that the oxidative stress-type cytotoxicity mechanism takes place. In addition, the cytotoxicity of nitroaromatics is also partly prevented by an inhibitor of NQO1, dicumarol. The cytotoxicity of the amino metabolites of TNT is also partly prevented by α- naphthoflavone and isoniazide, which points to the involvement of cytochromes P-450 in their activation. In general the cytotoxicity of nitroaromatics in splenocytes increases with an increase in their single-electron reduction potential, Eζ . This points to the prevailing mechanism of the oxidative stress-type cytotoxicity. The obtained structure-activity relationship and the studies of other mammalian cell lines showed that the immunotoxic potential of nitroaromatic explosives may decrease in the order tetryl ≥TNT ≥ hydroxylamino metabolites of TNT > amino and diamino metabolites of TNT.

Non-enzymatic glycosylation (glycation) is a spontaneous set of reactions between reducing sugars and free amino groups in proteins or other biomolecules leading to the formation of fluorescent and coloured compounds known as advanced glycation end products (AGEs). AGEs cause structural changes of key proteins in humans, and therefore they are related with a number of physiological processes and diseases such as aging, atherosclerosis, cataract, arthritis, Alzheimer’s disease. Two main strategies have been employed to prevent the formation of AGEs: a) low carbohydrate diet and b) pharmacological intervention. The latter includes treatment with reactive compounds which might be either sugar competitors (type A), carbonyl traps (type B) or free radical trapping antioxidants (type C). Acetylsalicylic acid (ASA, aspirin) is a good example of sugar competitor capable of inhibiting glycation by acetylating ε-amino groups of lysine residues in proteins. Taking into consideration the inhibiting effect of ASA on glycation we designed to study the antiglycation activity of other acetyl group-containing compounds (acetamides and acetyl esters) using the lysine-rich protein histone H1 as a model. The glycation of the histone H1 was carried out by either fructose or a complex mixture of glycating agents obtained from E. coli and monitored by fluorescent spectroscopy, SDS-PAGE and measurement of the content of reactive carbonyl groups in the target protein. Our results showed that the inhibitory effect of phenyl acetate, acetanilide, 4-acetamidophenylacetic acid and isopropenyl acetate was comparable to that of ASA. Based on the obtained results we conclude that these compounds act as free radical scavengers protecting proteins from the damaging effect of reactive oxygen species produced during the formation of AGEs.

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based on spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83± 1.96) μm for formylated bacterial tripeptide (fMLP) stimulation and (11.84±1.62) μm for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.

6-Alkylsalicylic acids inhibit the linoleic acid peroxidation catalyzed by soybean lipoxygenase- 1 (EC 1.13.11.12, type 1) competitively and without pro-oxidant effects. This activity is largely dependent on the nature of their alkyl side chains. Inhibitory activities of anacardic acids, viz. 6-pentadec(en)ylsalicylic acids, isolated from the cashew Anacardium occidentale, were initially used for comparison because their aromatic head portions are the same. Consequently, the data should be interpreted to mean that changes in the hydrophobic side chain tail portions of the molecules evaluated correlate with the specific activity determined.

We have tested acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of nineteen essential oils obtained from cultivated plants, namely one from Anethum graveolens L. (organic fertilizer), two from Foeniculum vulgare Mill. collected at fullymature and flowering stages (organic fertilizer), two from Melissa officinalis L. (cultivated using organic and chemical fertilizers), two from Mentha piperita L. and M. spicata L. (organic fertilizer), two from Lavandula officinalis Chaix ex Villars (cultivated using organic and chemical fertilizers), two from Ocimum basilicum L. (green and purple-leaf varieties cultivated using only organic fertilizer), four from Origanum onites L., O. vulgare L., O. munitiflorum Hausskn., and O. majorana L. (cultivated using organic fertilizer), two from Salvia sclarea L. (organic and chemical fertilizers), one from S. officinalis L. (organic fertilizer), and one from Satureja cuneifolia Ten. (organic fertilizer) by a spectrophotometric method of Ellman using ELISA microplate-reader at 1 mg/ml concentration. In addition, a number of single components widely encountered in most of the essential oils [γ-terpinene, 4-allyl anisole, (-)-carvone, dihydrocarvone, (-)-phencone, cuminyl alcohol, cumol, 4-isopropyl benzaldehyde, trans-anethole, camphene, iso-borneol, (-)-borneol, l-bornyl acetate, 2- decanol, 2-heptanol, methyl-heptanol, farnesol, nerol, iso-pulegol, 1,8-cineole, citral, citronellal, citronellol, geraniol, linalool, α-pinene, β-pinene, piperitone, iso-menthone, menthofurane, linalyl oxide, linalyl ester, geranyl ester, carvacrol, thymol, menthol, vanilline, and eugenol] was also screened for the same activity in the same manner. Almost all of the essential oils showed a very high inhibitory activity (over 80%) against both enzymes, whereas the single components were not as active as the essential oils.

Application of 100 μm aspterric acid (AA), a pollen growth inhibitor, with different concentrations of indole-3-acetic acid (IAA) results in the recovery of normal pollen development of Arabidopsis thaliana. Treatment with 100 μm AA plus 5 mm IAA significantly induced the normal seed production. Treatment with 100 μm N-1-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor, did not reduce the pollen growth but inhibited seed production. 100 μm NPA plus 5 mm IAA did not induce any seed production. The endogenous level of IAA in stems and leaves of A. thaliana treated with 100 μm AA was similar to that of the untreated control. In contrast to AA treatment, the IAA level by the treatment with 100 μm NPA was about twice as much as that of the untreated control. These results suggest that AA affects the Arabidopsis reproductive growth without inhibiting IAA biosynthesis and transport.

A screening test on 29 microorganisms for transformation of xanthohumol led to the selection of twelve fungal strains. One of them, Beauveria bassiana AM278, converted xanthohumol into a glucosylated derivative. This product was identified as xanthohumol 4’-O-β-D-4’’’- methoxyglucopyranoside.

Microbial transformation of glabratephrin, the major isolated compound from Tephrosia purpurea, afforded pseudosemiglabrin. The formation of the transformed compound seems to be performed via ring opening-closure of a five-membered ring causing transformation from a spiro into a fused system. The structure of the transformed compound was determined by comprehensive NMR studies, including DEPT, COSY, HMQC, NOE and MS.

The biosynthetic origins of the carbon skeleton in graphenone were verified by feeding the culture of spore-derived mycobionts of the lichen Graphis handelii with sodium [1- 13 C]- acetate, sodium [1,2- 13 C 2 ]-acetate, sodium [2- 13 C]-pyruvate, [1,2,3- 13 C]-glycerol, [ 13 CH 3 ]- methionine and sodium [1,4- 13 C 2 ]-succinate.

Changes in peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were studied during Chimonanthus praecox seed maturation. According to our findings the protein content increased steadily from 8 to 12 weeks after flowering, and thereafter decreased significantly. Similarly, SOD and POD activities increased gradually up to 12 weeks after flowering and then declined. PAL activity declined gradually during seed maturation. CAT activity, however, showed no changes during seed maturation. By means of polyacrylamide gel electrophoresis (PAGE), SOD and POD isoenzymes were observed during seed maturation. The staining intensities of SOD and POD isoenzymes correlated well with SOD and POD activities as obtained by an assay in solution. These findings suggest that POD, SOD and PAL may be involved in the growth and development during Chimonanthus praecox seed maturation.

Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called ‘housekeeping genes’ are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), α-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO 2 . In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.

The effect of drought on the chickpea variety ILC 3279 was investigated at the vegetative stage. After 20 days from sowing, the plants subjected to drought stress for 3, 5 and 7 days imposed by withholding water were permitted to recover by rewatering for 2 days after 3, 5 and 7 days of drought. Shoot elongation, leaf production, fresh and dry biomass reduced while MDA and proline accumulation increased with extended duration of stress. The plants stressed for 3 days exhibited a rapid drop in their relative and absolute water contents. The quantum efficiency of PSII open centres in the dark-adapted and light-saturated state, excitation energy trapping of PSII and electron transport rate decreased significantly from the 5 th day to the end of the drought treatments. Plants drought-stressed for 7 days brought about a marked increase in non-photochemical energy dissipation and a marked decline in photochemical quenching. After rewatering all chlorophyll a fluorescence characteristics except for F M completely recovered and reached the control values. Under 5 and 7 days of drought, the anthocyanin content increased gradually while the total chlorophyll content of leaves declined compared to the controls. The total carotenoid content remained unchanged during the experiments. The antioxidant enzyme response to drought treatments was quite variable. The total SOD activity upregulated with increasing duration of stress. On the other hand, the total APX activity was significantly higher only on the 7 th day while the total POD activity increased from the 5 th day. Differences in the total GR activity of treated groups were not statistically significant compared to their controls throughout the treatments. The present results indicate that the chickpea variety ILC 3279 withstands severe drought with its upregulated protective mechanisms at the vegetative stage.

Origanum onites is an economically important medicinal plant with high essential oil content. Lack of an appropriate DNA isolation procedure is a limiting factor for any molecular study of this plant. We have used a protocol for genomic DNA isolation based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method involves mortar grinding of leaf tissue, modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone, and successive isoamyl alcohol/chloroform extractions. The yield was approx. 20 μg DNA per 200 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in restriction digests, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) reactions. This extraction method should facilitate the molecular analysis of Origanum chemotypes.

Gasoline-contaminated soil from Isfahan, Iran was selected to isolate a bacterium capable of desulfurizing dibenzothiophene (DBT). The isolated strain was named R1 and identified as Rhodococcus erythropolis through biochemical tests as well as sequencing of 16S rRNA gene. This strain could efficiently produce 2-hydroxybiphenyl (HBP) from DBT via the 4S metabolic pathway. The highest HBP amount was produced at 2 mm DBT with addition of glucose (10 g l -1 ), ethanol (3 g l -1 ), glycerol (2 g l -1 ) or succinate (10 g l -1 ) as carbon sources at pH 7. Highest respiration and growth rates were observed by microplate titration on 0.1 mm HBP, and addition of 0.2 mm HBP to glucose (1 g l -1 ) and DBT (0.3 mm) could inhibite the respiration of the isolate. The isolated strain could grow up to 0.4 mm of HBP when it is used with mineral sulfur as sole sulfur source. To the best of our knowledge this is the first report on a microtiter assay for the production and utilization of HBP by Rhodococcus.

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-α, IFN-βand IFN-γ on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-α had a stronger inhibitory effect than IFN-γ. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a simple and inexpensive method for separating saccharides. Oligosaccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and the reductive amination reactions were essentially complete after approximately 16 h under the given experimental conditions. Saccharide-ANTS adducts were then separated by electrophoresis on 32% C ACR /2.4% C BIS polyacrylamide gel at alkaline pH. This technique doesn’t require sophisticated instrumentation and highly trained personnel.

A field test carried out in an industrial greenhouse in Lithuania revealed the attractiveness of synthetic methyl salicylate (MeSa) towards an economically important leafmining tomato pest, Liriomyza bryoniae (Kaltenbach) (Diptera, Agromyzidae). The behavioural reaction of the flies depended very much on the simultaneous presence of both olfactory and visual stimuli. The attractiveness depended on the colour of a sticky trap: MeSa attracted significantly more flies (ca. 2.2 times) when placed in yellow traps than in aluminium foil colour ones, when catches in such traps were compared to a corresponding control. L. bryoniae is the first species within the Agromyzidae family attracted by MeSa. The attractant was attributed to kairomones, as the compound is known as a plant-produced volatile. MeSa can be an effective extra-tool for increasing the attractiveness of traps. It should be evaluated in future whether such trap/bait combination is effective for the mass trapping of L. bryoniae leafminers in greenhouses (closed area).

To characterize the role of hen eggs used for analyzing the insect Mylabris phalerata a traditional Chinese medicine for curing liver cancer, egg white was separated from the whole egg and added to M. phalerata. The volatile compounds found in egg white when added to M. phalerata and therefore in M. phalerata itself were analyzed by GC. Cantharidin was detected both in egg white added to M. phalerata and M. phalerata itself. Egg white can be used as a favourable solvent for extracting the active ingredient of M. phalerata.

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L -1 sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 ± 0.6) mg g -1 dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 ± 0.15) mg g -1 dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.