Volume 62, Issue 5-6

The present study provides an update on the betaxanthin (bx) compositions of red and yellow beetroots, yellow-coloured Swiss chard petioles, and yellow-orange cactus pear. Applying RP-HPLC coupled with positive ion electrospray mass spectrometry and by comparison with UV-vis and mass spectrometric characteristics as well as retention times of semisynthesized reference compounds, 24 betaxanthins were identified in red and yellow beetroot hypocotyls. Twenty-five and thirteen betaxanthins were present in yellow Swiss chard petioles and the cactus pear cultivar ‘Gialla’, respectively. Ethanolamine-bx and threonine-bx were found to be novel betaxanthins in Chenopodiaceae representatives, which to the best of our knowledge have not been reported as genuine pigments so far. Furthermore, aspartic acidbx (miraxanthin II), lysine-bx, and methionine-bx, hitherto found in other families, were identified in the Chenopodiaceae for the first time. Additionally, tyrosine-bx (portulacaxanthin II) and tryptophan-bx have not been earlier reported to occur in the Cactaceae. These findings provide valuable phytochemical information and may be useful for a better understanding of the functional properties of betaxanthins in plants.

The methanol extract of the whole parts of Tribulus macropterus Boiss. (family Zygophyllaceae) showed cytotoxic activity against a human tumour cell line (hepatocyte generation 2, HepG2) (IC 50 = 2.9 μg/ml). The n-butanolic fraction obtained from successive fractionation of the methanolic extract exhibited activity against HepG2 (IC 50 = 2.6 μg/ml). Therefore, this fraction was subjected to separation using different chromatographic techniques. Five compounds, 1-5, were isolated and identified as: (22S,25S)-16β,22,26-trihydroxy-cholest-4- en-3-one-16-O-β-D-glucopyranosyl-(1→3)-β-D-xylopyranoside (1), (22S,25S)-16β,22,26-trihydroxy- cholest-4-en-3-one-16-O-β-D-glucopyranosyl-(1→3)-β-d-glucopyranoside (2), sucrose (3), d-pinitol (4) and 3β-hydroxy-5α-pregn-16(17)en-20-one-3-O-β-D-xylopyranosyl-(1→2)- [β-d-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside (5) on the basis of spectroscopic and chemical data. The three steroidal compounds 1, 2 and 5 were also tested against the same cell line HepG2 and their IC 50 values were 2.4, 2.2 and 1.1 μg/ml, respectively.

From the aerial parts of Anthemis tinctoria L. subsp. tinctoria var. pallida DC. (Asteraceae), one new cyclitol glucoside, conduritol F-1-O-(6′-O-E-p-caffeoyl)-β-d-glucopyranoside (1), has been isolated together with four flavonoids, nicotiflorin (2), isoquercitrin (3), rutin (4) and patulitrin (5). The structures of the isolated compounds were established by means of NMR, MS, and UV spectral analyses. Methanolic extract and pure isolated compounds were examined for their free radical, scavenging activity, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free stable radical, and for their inhibitory activity toward soybean lipoxygenase, using linoleic acid as substrate. Compounds 1 and 5 showed a strong scavenging effect in the DPPH radical assay. In addition 5 also exhibited high inhibitory activity on soybean lipoxygenase.

Phytochemical investigation of Cassia petersiana Bolle leaves afforded four new compounds, including two chromone derivatives, 7-acetonyl-5-hydroxy-2-methylchromone (petersinone 1, 1) and 7-(propan-2ʹ-ol-1ʹ-yl)-5-hydroxy-2-methylchromone (petersinone 2, 2), two benzoic acid derivatives, 5-methyl-3-(propan-2ʹ-on-1ʹ-yl) benzoic acid (petersinone 3, 3) and 5-(methoxymethyl)-3-(propan-2ʹ-ol-1ʹ-yl) benzoic acid (petersinone 4, 4), and glyceryl- 1-tetracosanoate (6), in addition to the known compound sistosterol-3-β-ᴅ-glycoside (5). The structures of these compounds were determined by comprehensive NMR studies, including DEPT, COSY, HMQC, HMBC, MS and IR. Compounds 1, 2, 5 and 6 were tested for antioxidant, anti-cancer and immunostimulatory properties. The biological investigations indicated that compound 6, among others, possessed the highest anti-cancer activity against hepatocellular carcinoma, immunoproliferative activity via induction of T-lymphocytes and macrophage proliferation, anti-inflammatory activity as indicated by NO inhibition, and antioxidant activity against DPPH radicals. Moreover, compound 5 was the most effective cytotoxic compound against breast carcinoma and stimulated a consistent immunoproliferative effect on lymphocytes and macrophages combined with strong NO inhibitory activity, while compound 1 was promising as immunoproliferative agent and may act as anti-inflammatory agent as a consequence of its NO inhibitory activity.

Extracts from Alternanthera maritima are used in Brazilian folk medicine for the treatment of infectious and inflammatory diseases. Bioassay-guided fractionation of A. maritima aerial parts yielded an ethanolic crude extract, its butanolic fraction and seven isolated flavonoids (two aglycones, two O-glycosides and three C-glycosides) with antioxidative activity. The ability of these samples to scavenge enzymatically generated free radicals (luminol-horseradish peroxidase-H 2 O 2 reaction) and inhibit reactive oxygen species (ROS) production by opsonized zymosan-stimulated human neutrophils (PMNLs) was evaluated by chemiluminescence methods. In both assays, the butanolic fraction was significantly more active than the ethanolic crude extract, the flavonoid aglycones had high inhibitory activities and the Cglycosylated flavonoids had no significant effect even at the highest concentration tested (50 μmol/L). However, the O-glycosylated flavonoids inhibitory effects on chemiluminescence were strongly dependent on the chemical structure and assay type (cellular or cell-free system). Under the conditions tested, active samples were not toxic to human PMNLs.

Crude ethanolic extract, lignoid fraction and the purified compound yangambin were obtained from Ocotea duckei (Lauraceae) and their antileishmanial activity was tested against promastigote forms of Leishmania chagasi and Leishmania amazonensis cultivated in Schneider medium, supplemented with 20% of fetal bovine serum. All substances presented antileishmanial activity with IC 50 values of 135.7 μg/mL for the crude ethanolic extract, 26.5 μg/mL for the lignoid fraction and 49.0 μg/mL for yangambin on L. chagasi. For L. amazonensis the IC 50 values were 143.7 μg/mL, 48.2 μg/mL and 64.9 μg/mL for the crude ethanolic extract, the lignoid fraction, and the purified compound yangambin, respectively. The crude ethanolic extract, lignoid fraction, and yangambin caused an inhibition higher than Glucantime“, a reference drug used for the treatment of leishmaniasis.

The dichloromethane extract of Calea uniflora afforded a mixture of two novel chromanones, uniflorol-A (1) and uniflorol-B (2), and one known chromanone, 2,2-dimethyl-6- (1-hydroxyethyl)-chroman-4-one (3). The structures of these compounds were determined by spectroscopic methods. Biological activity of the compounds against Leishmania major promastigotes was evaluated. Mixture of the novel chromanones 1 and 2 showed significant growth inhibition of the parasite in the micrograms per milliliter range.

Pathogenesis of chronic inflammatory diseases is associated with excessive elastase release through neutrophil degranulation. In the present study, inhibition of human neutrophil degranulation by four flavonoids (myricetin, quercetin, kaempferol, galangin) was evaluated by using released elastase as a biomarker. Inhibitory potency was observed in the following order: quercetin > myricetin > kaempferol = galangin. Quercetin, the most potent inhibitor of elastase release also had a weak inhibitory effect on the enzyme catalytic activity. Furthermore, the observed effects were highly dependent on the presence of a catechol group at the flavonoid B-ring. The results of the present study suggest that quercetin may be a promising therapeutic agent in the treatment of neutrophil-dependent inflammatory diseases.

The antifeedant effect of six cacalolides and six eremophilanolides was tested against the herbivorous insects Spodoptera littoralis, Leptinotarsa decemlineata, and Myzus persicae. The test compounds included several natural products isolated from Senecio madagascariensis (14-isovaleryloxy-1,2-dehydrocacalol methyl ether, 4), S. barba-johannis (13-hydroxy-14-oxocacalohastine, 5; 13-acetyloxy-14-oxocacalohastine, 6) and S. toluccanus [6-hydroxyeuryopsin, 7; 1(10)-epoxy-6-hydroxyeuryopsin, 9; toluccanolide A, 11] and the derivatives cacalol methyl ether (1); cacalol acetate (2); 1-acetyloxy-2-methyloxy-1,2,3,4-tetradehydrocacalol acetate (3); 6-acetyloxyeuryopsin (8); 6-acetyloxy-1(10)-epoxyeuryopsin (10), and toluccanolide A acetate (12). Compound 11 and its derivative 12 exhibited moderate antifeedant activity against S. littoralis; 2, 7D10, and 12 showed strong activity against L. decemlineata, while the aphid M. persicae was moderately deterred in the presence of compounds 1, 4, 8, 10, and 12. The phytotoxic activity of 1D12 on Lactuca sativa was also evaluated. Compounds 2 and 4D 12 moderately inhibited seed germination at 24 h, while compounds 1D4, 6, 9, and 10 had a significant inhibition effect on L. sativa radicle length (over 50%).

Phytotoxicity of parthenin, a sesquiterpene lactone, was evaluated against four weedy species (Amaranthus viridis, Cassia occidentalis, Echinochloa crus-galli, and Phalaris minor) through a series of experiments conducted under laboratory or greenhouse conditions to assess its herbicidal potential. Under laboratory conditions, parthenin (0.5 - 2 mm) severely reduced seedling growth (root and shoot) and dry weight of test weeds. However, the effect was greater on root growth. Parthenin (1 mm) suppressed the mitotic activity in the onion root tip cells that could possibly be responsible for the reduction in seedling growth. Both pre- and post-emergent application of parthenin caused a significant loss of chlorophyll pigments and affected photosynthesis. Parthenin (≥1 mm) caused an excessive electrolyte leakage in the plant tissues which was light-dependent. The root inhibition was associated with swelling and blackening of the root tip, shriveling and damage to the epidermal tissue and non-formation of root hairs. The study concludes that parthenin possesses weed-suppressing potential (both pre- and post-).

Leishmania (V.) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against N α -benzoyl-l-arginine ethyl ester hydrochloride and N α -p-tosyl-l-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.

The presence of various enzyme forms with terminal action pattern on pectate was evaluated in a protein mixture obtained from parsley roots. Enzymes found in the soluble fraction of roots (juice) were purified to homogeneity according to SDS-PAGE, partially separated by preparative isoelectric focusing and characterized. Three forms with pH optima 3.6, 4.2 and 4.6 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates) while the form with pH optimum 5.2 was a typical exopolygalacturonase [EC 3. 2.1.67] with relatively fast cleavage of polymeric substrate. The forms with pH optima 3.6, 4.2 and 5.2 were released from the pulp, too. The form from the pulp with pH optimum 4.6 preferred higher oligogalacturonates and was not described in plants previously. The production of individual forms in roots was compared with that produced by root cells cultivated on solid medium and in liquid one.

Diterpenoids in higher plants are biosynthesized from isoprene units obtained from two distinct pathways: the mevalonate pathway and the deoxyxylulose phosphate pathway. The metabolic partitioning of both pathways in plant species is dependent upon the type of culture. In order to study the diterpenoid biosynthesis in Croton stellatopilosus cell culture, callus culture was firstly induced from C. stellatopilosus young leaves in Murashige and Skoog (MS) medium in the presence of 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. The suspension culture was further induced from its callus in the same medium without gelling agent. Detection of diterpenoid accumulation by gas chromatography-mass spectrometry revealed that a cell culture could accumulate a low amount of geranylgeraniol (GGOH) and a high content of fatty acids and phytosterols. To improve the GGOH production, the culture conditions were optimized by medium manipulation in terms of hormonal factors. The growth rates of cell cultures were similar in all kinds of media. The GGOH production curve indicated that GGOH plays an important role as a primary metabolite in the cell culture. The optimum medium for GGOH production was MS medium supplemented with 2.0 mg/l 2,4-D and 2 mg/l BA that could produce GGOH with a yield of 1.14 mg/g FW.

Enantioselective hydrolysis of bromo- and methoxy-substituted 1-phenylethanol acetates was conducted using comminuted carrot (Daucus carota L.) and celeriac (Apium graveolens L. var. rapaceum) roots. Hydrolysis of the acetates led to alcohols, preferentially to R-(+)- enantiomers. Efficiencies of both reactions - hydrolysis of the acetates with an electrondonating methoxy group and oxidation of the resulting alcohols - increased in the following order: ortho < meta < para. The presence of an electron-withdrawing bromine atom in the aromatic ring had the opposite effect. Oxidation of alcohols with both types of substituents in the aromatic ring showed that location of a substituent had stronger impact on the oxidation rate than its electronic properties.

“NYB” is a chlorophyll-less barley mutant, which grows relatively slow and unhealthily. The effects of water stress on photosystem II (PSII) of NYB and its wild type (WT) were investigated. Unexpected results indicated that the mutant was more resistant to water stress, because: PSII core proteins D1, D2 and LHCII declined more in WT than in NYB under water stress, and the corresponding psbA, psbD and cab mRNAs also decreased more dramatically in WT; CO 2 assimilation, stomatal conductance, maximum efficiency of PSII photochemistry (Fv/Fm), efficiency of excitation energy capture by open PSII reaction centres (Fv’/Fm’), quantum yield of PSII electron transport (φ PSII ) and DCIP photoreduction in NYB were less sensitive to water stress than in WT, although the non-photochemical quenching coefficient (q N ) and the photochemical quenching coefficient (q P ) were almost the same in NYB and WT. Effective chlorophyll utilization and improved PSII protein formation in the mutant may be the reason for the enhanced stress resistance. Other possible mechanisms are also discussed.

The effect of water deficit on flavonoid production and physiological parameters characteristic for oxidative stress were studied in a cell suspension culture of Glycyrrhiza inflata Batal to investigate its drought tolerance. The result indicated that appropriate water deficit enhanced biomass accumulation of 27.1 g L -1 and flavonoid production of 151.5 mg L -1 , which was about 2-fold and 1.5-fold of the control, respectively. But it decreased the water content. Drought stress led to hydrogen peroxide accumulation more than in the control. Moreover, under drought conditions, malondialdehyde content, the activities of catalase and peroxidase increased to a greater extent than the control, and each reached a maximum value of 91.3 μmol g -1 dry weight, 85.6 U and 1951 U g -1 dry weight per min, which was 1.5-, 1.7- and 3.7-fold of the control, respectively. All above showed that appropriate water deficit could activate the antioxidative defense enzymes system to maintain stability in plants subjected to drought stress. On the contrary, the activity of phenylalanine ammonia lyase of the control increased in company with the biosynthesis of flavonoids, which indicated that phenylalanine ammonia lyase might play an important role in the path of the biosynthesis of flavonoids.

We have compared the effect of toxic Cu and Cd concentrations on growth, metal accumulation, and chloroplast ultrastructure of willow (Salix purpurea L.) and reed [Phragmites australis (Cav.) Trin. ex Steud.]. After a 10-day treatment, both species have tolerated to some extent the lowest concentration of both metals; however, plant growth was strongly reduced at the highest Cu and Cd concentrations. These plants could be described as Cutolerant at the lowest concentration tested, showing a higher tolerance index in reed than in willow; in contrast, willow exhibited higher tolerance against Cd. Both plants appeared to be moderate root accumulators of Cu and Cd. Ultrastructural studies revealed special features that can provide some protection against heavy metals stress, such as ferritin aggregates in the stroma. In addition, Cu and Cd induced distortion of thylakoids, reduction of grana stacks, as well as an increased number and size of plastoglobuli and peripheral vesicles.

This work is a continuation of earlier research concerning the influence of tin compounds on the dynamic properties of liposome membranes produced with lecithin hen egg yolks (EYL). The experiments were carried out at room temperature (about 25 ∞C). Four tin compounds were chosen, including three organic ones, (CH 3 ) 4 Sn, (C 2 H 5 ) 4 Sn and (C 3 H 7 ) 3 SnCl, and one inorganic, SnCl 2 . The investigated compounds were admixed to water dispersions of liposomes. The content of the admixture changed within the range 0 mol-% to 11mol-% in proportion to EYL. Two spin probes were used in the experiment: 2,2,6,6-tetramethylpiperidine- 1-oxyl (TEMPO) and 2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxyl (16-DOXYL-stearic acid), which penetrated through different areas of the membrane. It was found that tin compounds containing chlorine were the most active in interaction with liposome membranes. In the case of (C 3 H 7 ) 3 SnCl, after exceeding 4% admixture content, an additional line appeared in the spectrum of the TEMPO probe which can be a result of formation of domain structures in the membranes of the studied liposomes. Compounds containing chlorine are of ionized form in water solution. The obtained results can thus mean that the activity of admixtures can be seriously influenced by their ionic character. In case of an admixture of non-ionic compounds the compound with a longer hydrocarbon chain displayed a slightly stronger effect on the spectroscopic parameters of the probes.

Bacterial biofilms which are responsible for a number of diseases are very difficult to control effectively because of their high resistance to antibiotics and the host defence system. The use of natural products decreasing or preventing initial adhesion of bacteria and biofilm formation is one of the alternative therapeutic strategies taken into consideration. We ask the question, whether a crude extract from the cell wall of Saccharomyces cerevisiae (mannoprotein), which possesses surfactant activity, may be used as inhibitor of Staphylococcus aureus and S. epidermidis biofilm development. By using the “bactericidal spot assay” it was demonstrated that mannoprotein had no direct antibiotic activity against the tested strains. The influence of this extract on initial adhesion, biofilm formation and dispersal of preformed biofilms was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. In this assay, live bacteria with an active electron transport system reduce the tetrazolium salt to a water-soluble purple formazan product, and optical density reading (A 550 ) values are directly dependent on their cell numbers. Yeast-derived surfactant, when adsorbed in the microplate wells or present in the medium, was effective both in decreasing the initial deposition of staphylococci and in reducing the amount of growing biofilm, quantitated after 24 h of co-incubation with the bacteria. It also changed the parameters of biofilm morphology analyzed by PHLIP - the confocal laser scanning microscopy image quantification package. Mannoprotein also accelerated the detachment of mature staphylococcal biofilms, preformed in optimal conditions. It was concluded that mannoprotein anti-biofilm action reflects its influence on cell surface hydrophobicity.

Hairy root cultures from red beet (Beta vulgaris L.), which could be used for the commercial production of biologically active betalain pigments, were cultivated in a 3 L bubble column bioreactor in batch mode with various rates of air supply. Both the growth of the roots and betalain volumetric yields were highest (12.7 g accumulated dry biomass/L and 330.5 mg/ L, respectively) with a 10 L/h (0.083 vvm) air supply. The air flow rate also influenced the betacyanins/betaxanthins ratios in the cultures. Growth and betalains production were then examined in two fed-batch regimes (with a 10 L/h air supply), in which nutrient medium was fed just once or on five occasions, designated FBI and FBII, respectively. The root mass accumulation was increased in the FBI feeding regime (to 13.3 g accumulated dry biomass/ L), while in FBII the betalains content was ca. 11% higher (15.1 mg betacyanins/g dry weight and 14.0 mg betaxanthins/g dry weight) than in the most productive batch regime. Data on the time course of the utilization of major components in the medium during both operational modes were also collected. The implications of the information acquired are discussed, and the performance of the hairy roots (in terms of both growth and betalains production) in the bubble column reactor and previously investigated cultivation systems is compared.

The biomass production of Cymbopogon citratus shoots cultivated in bioreactors according to the temporary immersion (TIS) principle was assessed under different growth conditions. The effect of gassing with CO 2 -enriched air, reduced immersion frequency, vessel size and culture time on total phenolic and flavonoid content and free radical scavenging effect of the methanolic extracts was measured. From the TIS-culture of C. citratus, seven compounds were isolated and identified as caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3), p-hydroxybenzoic acid (4), p-hydroxybenzoic acid 3-O-β-d-glucoside (5), glutamic acid (6) and luteolin 6-C-fucopyranoside (7). The occurrence of compounds 1-7 and their variability in C. citratus grown under different TIS conditions was determined by HPLC. The free radical scavenging effect of the methanolic extract and compounds was measured by the discoloration of the free radical 1,1- diphenyl-2-picrylhydrazyl (DPPH). The main metabolites in 6- and 8-week-old cultures, both in 5 and 10 l vessels, were chlorogenic acid (2) (100D113 mg%) and neochlorogenic acid (3) (80 - 119 mg%), while in the cultures with CO 2 -enriched air and reduced immersion frequency the main compound detected in the extracts was glutamic acid (6) (400 and 670 mg% for the green and white biomass and 619 and 630 mg% for the green and white biomass, respectively). The most active compounds, as free radical scavengers, in the DPPH discoloration assay were caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3) and the flavonoid luteolin 6-C-fucopyranoside (7).

The induction of apoptosis by 66 alkaloids of the quinoline, quinolizidine, pyrrolizidine, isoquinoline, indole, terpene, tropane, steroid, purine, and piperidine type, of 9 cardiac glycosides, 11 non-protein amino acids and 10 further secondary metabolites was assayed in HL- 60 cell cultures and measured by quantification of the subdiploid DNA content by flow cytometry, detection of DNA fragmentation by gel electrophoresis, and cell morphology. Several alkaloids of the isoquinoline, quinoline, and indole type were active, whereas quinolizidine, tropane, pyrrolizidine, terpene and piperdine alkaloids were mostly inactive. The proapoptotic alkaloids can be characterized by their property to inhibit protein biosynthesis and their intercalation into DNA at the same time, or by their inhibition of microtubule formation. All cardiac glycosides, which are both membrane detergents and Na + ,K + -ATPase inhibitors, are potent apoptosis inducers. Also proapoptotic were a few non-protein amino acids, podophyllotoxin and the flavonoid quercetin.