Band 63, Heft 9-10

Roridula dentata and R. gorgonias, two South African plants that were formerly believed to be carnivorous, exhibit an extremely sticky exudate at the tips of secretory trichomes. Unlike the trapping mucilage of Droseraceae, it does not consist of acidic polysaccharides. The Roridula trapping glue was found to be a mutual solution of mainly dihydroxytriterpenoids, instead. All samples contain two isomers of ring A dihydroxyolean-12-enes and dihydroxyurs- 12-enes. The difference between the two species is the additional presence of taraxeradiol in the glue of R. gorgonias. The absolute chemical structures of the reported triterpenoids still need confirmation.

The variability of sorbicillin derivatives production by marine Trichoderma sp., isolated from the sponge Agelas dispar J., was studied using six different culture media to find a good medium for biomass production, particularly of trichodimerol, bislongiquinolide and bisvertinol. A simple and rapid convenient method for identification, quantification and validation of the sorbicillin derivatives in ethyl acetate extracts has been applied by using RPHPLC coupled with diode array detection.

Purpurone was isolated from the sponge Iotrochota sp. by bioactivity-guided fractionation. The compound showed antioxidant activity using the DPPH assay. The structure was established on the basis of NMR data and comparison with data reported.

In the context of the study on plant defensive compounds we have isolated the main alkaloid from Hippeastrum puniceum (Amaryllidaceae), 3-O-acetyl-narcissidine (1), and its biological activities tested against two divergent insect species and several plant species. 1 was isolated from the bioactive alkaloidal fraction of H. puniceum. Its chemical structure was established by spectroscopic analysis. The biological activity tests showed that 1 is an antifeedant against the polyphagous insect Spodoptera littoralis but not against the olyphage Leptinotarsa decemlineata. Furthermore, the root growth of Amaranthus hypochondriacus, Rottboellia cochinchinensis, Panicum maximum and Solanum lycopersicum was significantly affected by 1. These results suggest a plant protective role for H. puniceum alkaloids.

The aim of this study was to investigate the antibacterial activity of chitooligosaccharide, prepared by partial acid hydrolysis of chitosan, and of an aminoglycosylated derivative, prepared by reductive alkylation of the chitooligosaccharide, against E. coli and S. aureus.

The antimicrobial activity of ethanol and chloroform extracts of Pentanema divaricatum Cass. was studied using the conventional disk diffusion method. The extracts’ highest antimicrobial activity was observed against Aspergillus niger. Bioassay-guided fractionation of the crude extract by preparative thin layer chromatography (PTLC) showed one antimicrobial fraction which was especially effective against Aspergillus niger. By conventional spectroscopy the active fraction was identified as 4α,5α-epoxy-10α,14H-1-epi-inuviscolide. This compound represented the most potent antimicrobial candidate, with MIC values of < 25 μg/disk against A. niger strains and 200 μg/disk against Bacillus cereus and Staphylococcus aureus.

Calia secundiflora (Ortega) Yakovlev (Fabaceae) is considered a medicinal plant in Mexico but has scarcely been used because of the toxicity of its quinolizidine alkaloids. Several quinolizidine alkaloids have shown bactericidal, nematicidal, and fungicidal activities. The purpose of this study was to identify the alkaloids in the seeds and evaluate the activity of the organic extract on several phytopathogenic fungi and bacteria. An in vitro bioassay was conducted with species of the following phytopathogenic fungi: Alternaria solani, Fusarium oxysporum and Monilia fructicola; and of the following bacteria Pseudomonas sp., Xanthomonas campestris and Erwinia carotovora. Cytisine, lupinine, anagyrine, sparteine, N-methylcytisine, 5,6-dehydrolupanine, and lupanine were identified by liquid chromatography-mass spectrometry in the extract of seeds; the most abundant compound of the extract was cytisine. It was observed that the crude extract of Calia secundiflora was moderately active on bacteria and more potent on phytopathogenic fungi. In contrast cytisine showed the opposite effects.

Seven flavonoids were isolated from the butanol fraction of the methanolic extract of the aerial parts of Cynanchum acutum L. (Asclepiadaceae). All of which have been isolated for the first time from the genus Cynanchum. Their structures were established as quercetin 3-O-β-galacturonopyranoside (1), quercetin 7-O-β-glucopyranoside (2), tamarixtin 3-O-β-galacturonopyranoside (3), kaempferol 3-O-β-galacturonopyranoside (4), 8-hydroxyquercetin 3-O-β-galactopyranoside (5), tamarixtin 3-O-α-rhamnopyranoside (6), and tamarixtin 7-O-α-arabinopyranoside (7) on the basis of their chromatographic properties, chemical and spectroscopic data. The major isolated flavonoids 1, 2 and 3 were found to exhibit significant antioxidant and antidiabetic activities (by measuring blood glucose and insulin levels). This is the first report about the antioxidant and antidiabetic activities of compounds 1 - 3.

The phytotoxicity of the three major monoterpene constituents of the essential oil from leaves of Artemisia scoparia Waldst. & Kit. (redstem wormwood) was investigated. GC/GCMS analysis revealed that the essential oil (yield 0.84%) is a complex mixture containing 19 monoterpenes, 7 sesquiterpenes and 15 other compounds - aliphatic alcohols, ketones, aromatic hydrocarbons and esters. The three major monoterpenes were β-myrcene (30.2%), p-cymene (12.8%) and dl-limonene (12.4%). The essential oil and the three monoterpenes exhibited phytotoxicity and reduced germination, seedling growth, chlorophyll content and percent respiration of Avena sativa and Triticum aestivum in a dose-response manner. The inhibitory effect of monoterpenes was comparatively smaller than of the crude essential oil and β-myrcene was most toxic followed by p-cymene, whereas limonene was least toxic. The study suggests that A. scoparia oil and β-myrcene can be explored for phytotoxicity against weeds.

A number of 1,3,4-oxadiazole, 3 - 9, and 1,2,4-triazole derivatives, 13 - 15, were synthesized starting form the acid hydrazide 1. The 1,3,4-thiadiazole derivative 12 was prepared from the substituted phenylthiosemicarbazide derivative 11 by treatment with sulfuric acid. The aryl hydrazone derivatives 10a - c were synthesized by reaction of the hydrazide 1 with the corresponding ketones. The thioalkyl derivatives 16a - e were prepared by akylation of the thiol derivatives 3 and 13 with different alkylating agents. The newly synthesized compounds were tested for their anti-HBV activity and some of these compounds showed high antiviral activity

The present study describes the cytotoxic properties of a series of 15 cyclic imides observed against different endothelial cells and K562 leukemic cells. Initially, eight structurally unrelated compounds were evaluated against cultured bone marrow endothelial cells (BMEC) and human umbilical vein endothelial cells (HUVEC). Only two imides showed cytotoxic activity at 10 μm. In continuation of our screening, eight compounds, structurally related to the compound with the higher cytotoxic activity, were assayed against endothelial cells and the K562 leukemic cell line. All of these new compounds except two exhibited cytotoxic and antiproliferative activities at concentrations below 10 μm against BMEC and HUVEC, respectively. The K562 leukemia cell line was only affected by concentrations of 100 μm. Preliminary SAR analysis indicated that the cytotoxic activity of these compounds was related to the presence of a planar imide ring directly bound to an aromatic ring.

The in vitro cytotoxic potential of yangambin was evaluated. Yangambin is a pharmacologically active furofuran lignan obtained from the leaves of Ocotea duckei. It is the major compound from the lignoids fraction. Yangambin presented low cytotoxicity in all in vitro models analyzed. Its cytotoxicity to murine macrophages was measured by the Trypan blue dye exclusion test and MTT reduction assay, resulting in high CC 50 values of 187.0 μg/mL (383.3 μm) and 246.7 μg/mL (504.3 μm), respectively. The difference obtained in the inhibitory concentrations aforementioned can be explained, at least in part, by the different principles of the methods. While the MTT reduction assay evaluates the ability of yangambin to inhibit the activity of the mitochondrial enzyme succinate dehydrogenase, the Trypan blue dye exclusion test evaluates possible damages to the integrity of the cytoplasmic membrane which result in cell death. The capacity of yangambin to inhibit the sea urchin embryonic development showed that it has low antimitotic and teratogenic potential, once continued exposure of embryos to concentrations up to 500 μg/mL (1.025 μm) did not result in an inhibitory effect on the first egg cleavages. Such low in vitro cytotoxicity is correlated with the low acute toxicity previously studied. All these data, together with the various therapeutic properties of yangambin, make this lignan a promising one for a new drug.

Oxidation of p-coumarate at the ortho-position is a key step to form umbelliferone. A tracer analysis using 18 O 2 was performed in order to take information about the formation of umbelliferone in the root tissue of sweet potato. Mass fragmentation experiments revealed incorporation of an 18 O atom into the 1-position of umbelliferone. This result indicates that the lactone of umbelliferone is formed via ortho-hydroxylation of the p-coumarate unit using O 2 .

Hairy root cultures of Mitragyna speciosa were established by infection of Agrobacterium rhizogenes ATCC 15834 and maintained in McCown woody plant medium (WPM) supplemented with 0.5 mg/l naphthaleneacetic acid. The hairy roots were identified for the rooting genes loci of rolA and rolB by polymerase chain reaction. For studying the secondary metabolite production, the n-hexane extract of the hairy roots was prepared and the compounds were isolated by silica gel column chromatography, affording triterpenoids (ursolic acid and oleanolic acid) and phytosterols (β-sitosterol and stigmasterol). The shoots from the hairy root cultures were regenerated and differentiated to the plantlets. For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron. The mitragynine contents of 5-monthold regenerated plants and in vitro plantlets (germinated from seeds) were determined using the TLC-densitometric method. The regenerated plants contained (14.25 ± 0.25) mg/g dry wt mitragynine, whereas the in vitro plantlets contained (4.45 ± 0.09) mg/g dry wt.

A cell suspension culture of sunflower (Helianthus annuus), a producer of immunologically active polysaccharides, was cultivated in a 5-L stirred tank bioreactor, operated in batch mode. After some changes in the internal bioreactor design a stable growth of Helianthus cells was achieved and the accumulated biomass reached 15.2 g/L (only ~5% lower compared to the accumulated biomass in shake-flasks). Flow cytometry used for measuring the cell cycle parameters of suspended Helianthus cells did not reveal significant differences between shake-flasks and bioreactor cultivation modes. For both cultivation methods significant enhancement of the percentage of S-phase cells was observed at the beginning of the cultivation process. Concerning the metabolite production the maximum in exopolysaccharides was reached at day 9 of the cultivation period (1.9 g/L), while the highest amounts of α-tocopherol were accumulated at the beginning of the cultivation process (day 2 of the cultivation). These finding were related to the respective stress levels caused by the inoculation procedure. The kinetic parameters of growth and polysaccharide production as well as the time course of carbon source utilization were monitored and discussed.

The alternative pathway (AP) respiration of plants is a cyanide-resistant and non-phosphorylating electron transport pathway in mitochondria. Alternative oxidase (AOX) is the terminal oxidase of the AP and exists in plant mitochondria as two states: the reduced, noncovalently linked state or the oxidized, covalently cross-linked state. In the present study, the effects of 20 μm exogenous salicylic acid (SA) on both AP activity and AOX expression in mitochondria of tobacco (Nicotiana rustica L. cv. yellow flower) calli were investigated. The results showed that SA treatment enhanced the AP activity. During the process of SA treatment, the AP activity increased dramatically and achieved the peak value after 8 h of treatment. Then it declined until 16 h, and maintained a steady level between 16 and 24 h. Changes in both the total AOX protein level and the reduced state were in accordance with the AP activity, but the oxidized state changed differently. The aox1 gene transcript level also showed a similar change as the AP activity and AOX protein level. The induction of AOX expression by low concentrations of SA was inferred through a reactive oxygen species (ROS)-independent pathway. These results indicate that the enhancement of AP activity in response to SA is correlated to the expression of AOX, and the reduced, non-covalently linked state of AOX plays an important role during this process.

The activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) were found to increase in suspensioncultured Arabidopsis thaliana cells after 10-day-old stationary phase cells were transferred to fresh Murashige-Skoog medium. The activities of these enzymes peaked early in the exponential growth stage of the culture (day 4) and then decreased gradually. The transcript levels of six isoform genes of G6PDH (AtG6PD1 to AtG6PD6) and three of 6PGDH (At6PGD1 to At6PGD3) were monitored during the culture. Two distinct transcript accumulation patterns were observed. In type A, the level of transcripts increased rapidly one day after the cells were inoculated into the fresh medium, and then remained almost constant until the culture reached its stationary phase (day 7). In type B, the transcripts were accumulated transiently at the first day after cell inoculation, then promptly decreased. We also investigated the effect of phosphate (Pi)-starvation and recovery on the expression of these genes. For this, the early stationary phase cultures (day 7) were transferred to fresh Pi-free culture medium. During 7 days of phosphate starvation, no growth of cultures was observed, and the transcript levels of all G6PDH and 6PGDH isoform genes were reduced, apart from one G6PDH isoform gene, AtG6PD5, which was continuously expressed throughout Pi-starvation. Compared to the reduction of almost all isoform genes of G6PDH in Pi-starved cultures, the reduction of 6PGDH genes was less severe. We discuss the localization and possible role of individual isoform genes of G6PDH and 6PGDH in connection with published databases.

The time-course of expression of dbat and dbtnbt genes involved in the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation were investigated through a 64-day subculture interval of VI/M1 and VI/M2 Taxus baccata callus cultures. HPLC proved traces of baccatin III and an intracellular content of paclitaxel up to 90 μg/g DW. The steadystate of the respective gene transcripts was measured by quantitative real-time RT-PCR. The expression profile of dbat and dbtnbt genes was slightly different and varied within the subculture. The highest level of dbat expression was detected 24 h after inoculation followed by a decrease in both cultures. In contrast with dbat no substantially high expression of the dbtnbt gene after inoculation was observed. The impact of the conditions during inoculation on gene expression is discussed. Although the increase in transcriptional activity of both genes positively correlated with callus growth, the intracellular accumulation of paclitaxel varied during subculture with the maximum in the stationary (VI/M1) or at the end of the linear (VI/M2) phase. The increase of the steady-state mRNA level of the dbtnbt gene was followed by paclitaxel accumulation with a delay of approx. 28 (VI/M1) and 14 days (VI/M2).

The lipophilic exudates deposited on aerial parts of four species of Cassinia and twelve species of Ozothamnus (Asteraceae, Gnaphalieae) have been analyzed for the presence of flavonoid aglycones and some other phenolics. A total of 55 flavonoids were identified, including several rare flavonols. Flavonols are prevailing over flavones, and 8-O-substitution is dominant in both groups. Ozothamnus rosmarinifolius is exceptional in producing several coumarins. Four dihydrobenzofurans have also been identified from this species. Cluster analysis and principal coordinate analysis of the flavonoid data provide support for combining species of Cassinia and Ozothamnus into a single genus.

Trichoderma spp. has been identified as potential antagonist of Fusarium solani, which is causing dry root rot of Citrus. A random amplified polymorphic DNA (RAPD) marker was used to estimate the genetic variation among 17 isolates of Trichoderma. These isolates were characterized using 20 random primers of the OPM series, out of which 16 primers gave a total of 145 DNA fragments, showing 91.8% polymorphism. The genetic distance between each isolate was calculated, and cluster analysis was used to generate a dendrogram showing the relationship among them. The isolates grouped into two major clusters, the first major cluster consisted of TCT 14 , TCT 17 , TCT 13 , TCT 12 and TCT 16 . The remaining isolates in the second major cluster separated in two sub-clusters; the first cluster consisted of TCT4, TCT 10 , TCT 2 , TCT 3 , TCT 8 , TCT 6 , TCT 9 , and the second sub-cluster consisted of TCT 1 TCT 15 TCT 5 , TCT11, and TCT7. The similarity matrix indicated that TCT6 and TCT13 were genetically distinct as they showed only 22.6% similarity followed by TCT 5 and TCT 16 ; TCT 6 and TCT 16 (25%), while the isolates TCT 4 and TCT 10 were found to be genetically similar, as 66.7% similarity was observed between the isolates followed by 61.3% similarity between the TCT 2 and TCT 4 isolates.

A protocol was developed for the in vitro propagation of ginger (Zingiber officinale) cv. Suprava using dormant axillary buds from unsprouted rhizomes. The dormant axillary buds embedded in the rhizome nodes were induced to sprout when cultured on MS medium supplemented with 6-benzyladenine (BA) alone (1 - 6 mg/l) or with a combination of BA (1 - 6 mg/l) and indole-3-acetic acid (IAA) (0.5, 1 mg/l). In vitro sprouted buds were transferred to the multiplication medium containing various combinations of auxins and cytokinins. MS basal medium supplemented with BA (1 mg/l), IAA (1 mg/l) and adenine sulfate (100 mg/l) was found optimum for the in vitro multiplication of shoots producing (8.2 ± 0.2) shoots from a single explant within 30 days of culture. The multiplication rate remained unchanged in subsequent subcultures. Rooting of shoots occurred in the same multiplication media. Upon transfer of the in vitro culture to ex vitro in pots, 96% of plants survived and established successfully under natural conditions. Tissue culture-raised plantlets of ginger could be conserved in vitro through subculturing at an interval of 4 months. The genetic stability of micropropagated clones was evaluated at regular intervals of 6 months up to 24 months in culture using cytophotometric estimation of 4C nuclear DNA content and random amplified polymorphic DNA (RAPD) analysis. Cytophotometric analysis revealed a unimodal distribution of the DNA content with a peak corresponding to the 4C value (23.1 pg), and RAPD analysis revealed monomorphic bands showing the absence of polymorphism in all fifty regenerants analyzed, thus confirming the genetic uniformity among in vitro grown somaclones of Z. officinale. This study is of commercial significance as axillary bud explants are available throughout the year for initiating a fresh culture of the elite ginger cv. Suprava to be used as a source of true-to-type disease-free planting material thereby minimizing the adverse effect of repeated subculturing from the same explant source.

Eight compounds isolated from Indian barnyard millet have been identified as l-malic acid, trans-aconitic acid, (+)-isocitric acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, isocarlinoside, 2″-O-rhamnosylisoorientin, and 7-O-(2″-O-glucuronosyl)glucuronosyltricin, respectively. These compounds showed high antifeeding activity against brown planthopper only when they were combined.

In electroantennographic tests isosafrol, methyl salicylate, (±)-lavandulol, geraniol, (E)-anethol, and β-ionone evoked the largest responses from antennae of female or male Oxythyrea funesta (Coleoptera: Scarabaeidae, Cetoniinae) adult beetles. In field trapping tests in Hungary the 1: 1 blend of (±)-lavandulol and 2-phenylethanol attracted significantly more adult O. funesta than the single compounds. The addition of (E)-anethol, a previously described attractant for the species, was without effect. There was no difference in the responses of male or female beetles. The binary 2-phenylethanol/(±)-lavandulol bait described in this study is recommended for the use in traps of O. funesta for agricultural purposes.

The dorsal gland secretions of captive-reared male and female Chacoan peccaries (Catagonus wagneri) were analyzed by gas chromatography-mass spectrometry. C 8 -C 19 carboxylic acids, squalene, cholesterol, cholestanol, and cholest-7-en-3-ol were present in both males and females. Heptylbenzene, C 14 -C 18 methyl esters, and an isomer of springene were observed in males. C 15 -C 19 aldehydes were observed in females. The composition of the dorsal gland secretions of C. wagneri is compared to what has been reported for other peccaries (Tayassu spp.).

The aim of the present study was to assess if some flavonic compounds (quercetin, piceatannol and apigenin) and ascorbic acid could interfere with the Lf stimulatory effect on the erythrocyte function. Quercetin (1.5 μm) and piceatannol (30 μm) showed an additive effect on Lf stimulation of Na + /K + -ATPase when used together with Lf. The enhancement of Lf stimulation on Na + /K + -ATPase in the presence of flavonoids was probably due to their antioxidative properties and/or to their involvement in the erythrocyte signaling. None of the estimated flavonoids showed an effect on Lf stimulation of the lactate production. Quercetin itself enhanced the ATPase activity but did not affect the lactate formation. Apigenin (1.5 μm) enhanced reliably the lactate generation, but it did not exert any effect on the ATPase activity. High concentration of ascorbic acid (60 mm) did not change the Lf stimulatory effect on Na + /K + -ATPase, but decreased the Lf-specific-binding. A significantly strong inhibitory effect on the Lf-specific binding exerted the electron acceptors NAD + (2 mm) and FAD (2 mm). These effects concern most likely the competition with Lf for electron(s) which is (are) provided from the erythrocyte intercellular electron transport chain(s).

Cytochrome P450 (CYP) is a heme-containing enzyme superfamily metabolizing a wide variety of xenobiotics, including drugs and carcinogens. The majority of CYP genes are expressed in the liver, however, some CYP isoforms are also reported for a number of extra hepatic tissues. We analyzed Cytochrome P450-2A6, -3A5 and -4B1 mRNAs using realtime reverse-transcriptase polymerase chain reaction (RT-PCR) in a total of 21 homogenized prostate tissues with or without malignancy. We detected a consistent expression of CYP2A6 and CYP3A5 in all, and of CYP4B1 in some (11/21) of the samples at mRNA level. Neither the histopathological status nor the smoking habit of the individuals affected CYP4B1 expression. Our results reflect possible roles for these particular CYPs in therapy and protection of prostate tissue.