Band 60, Heft 11-12

From the aerial parts of Astragalus suberi L., Fabaceae, seven saponins were isolated. Based on spectral data (IR, 1 H and 13 C NMR and HR-FABMS), the structures were established as 3-O-(β-ᴅ-glucopyranosyl)-soyasapogenol B (1); 3-O-(β-ᴅ-glucuronopyranosyl)- soyasapogenol B (2); 3-O-[β-ᴅ-galactopyranosyl (1→2)-β-ᴅ-glucopyranosyl]-soyasapogenol B (3); 3-O-[α-ʟ-rhamnopyranosyl (1→2)-β-ᴅ-galactopyranosyl (1→2)-β-ᴅ-glucopyranosyl]- soyasapogenol B (4); 3-O-[α-ʟ-rhamnopyranosyl (1→2)-β-ᴅ-galactopyranosyl (1→2)-β-ᴅ-glucopyranosyl]-11-hydroxy-soyasapogenol B (5); 3-O-[α-ʟ-rhamnopyranosyl (1→2)-β-ᴅ-galactopyranosyl (152)-β-ᴅ-glucuronopyranosyl]-soyasapogenol B (6) and 3-O-[α-ʟ-rhamnopyranosyl (1→2)-β-ᴅ-galactopyranosyl (1→2)-β-ᴅ-glucuronopyranosyl]-complogenin (7). The isolated saponins exhibited antibacterial activities against Gram-positive and Gramnegative bacteria with minimum inhibitory concentration values > 100 μg/ml, antifungal activity against all the strains tested with minimum fungicidal concentration values between 25 and 50 μg/ml and inhibited the growth of Hep-2 (human carcinoma of larynx), with IC 50 values between 50 μg/ml (compounds 5-7) and 100 μg/ml (compounds 1-4), and Hela (human carcinoma of cervix) cell lines in culture with different IC 50 values [74 compound 7), 98 (compound 5) and 180 μg/ml (compounds 1-4 and 6)]

Antimicrobial activity of the essential oils obtained from leaves, root and the seeds of Diplotaenia damavandica Mozaffarian, Hedge & Lamond, an endemic plant to Iran, was determined against 10 microorganisms using the disk susceptibility test as well as measuring minimum inhibitory concentrations. The results showed that all three oils had antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. The essential oil from the leaves had the highest antimicrobial activity against all test microorganisms including the fungal strains. The essential oils compositions were analyzed and determined by GC and GC-MS. The oils analyses resulted in the identification of 16, 17 and 20 compounds representing 94.2%, 96.4% and 95.1% of the total oils, respectively. The main components of the leaf essential oils were (Z)-β-ocimene (21.6%), α-phellandrene (21.3%) and terpinolene (20%). Dill apiol (30.1%) and γ-terpinene (16.2%) were the main components of the root and seed essential oils, respectively.

Polyphenolic compounds such as eriocitrin, luteolin-7-O-rutinoside, diosmin, hesperidin, narirutin, isorhoifolin, rosmarinic and caffeic acids were identified in an aqueous extract (Ex) obtained from peppermint leaves (Menthae × piperitae folium). The content of polyphenols in Ex was as follows: eriocitrin 38%, luteolin-7-O-rutinoside 3.5%, hesperidin 2.9%, diosmin 0.8%, isorhoifolin 0.6%, narirutin 0.3%, rosmarinic acid 3.7% and caffeic acid 0.05%. The strongest antiradical activity (determined as DPPH • scavenging features) was observed for luteolin-7-O-rutinoside, eriocitrin and rosmarinic acid. Caffeic acid and hesperidin revealed a lower antiradical activity while isorhoifolin, narirutin and diosmin showed the lowest activity. The strongest anti-H 2 O 2 activity was observed for eriocitrin, a little lower for rosmarinic acid. The rate of hydrogen peroxide scavenging activity displayed by luteolin-7-O-rutinoside and caffeic acid was lower than that of rosmarinic acids. Hesperidin appeared to be a very weak scavenger of hydrogen peroxide. Almost no anti-H 2 O 2 activity was demonstrated for diosmin, narirutin and isorhoifolin. Among examined flavonoids, the strongest antiradical and anti-H 2 O 2 activity was shown for compounds with two hydroxy groups bound to the Bring in ortho position in relation to each other. Replacement of one hydroxy group in the Bring with a methoxy group or removing one hydroxy group leads to decrease of antiradical and anti-H 2 O 2 activity of flavonoids. Our results suggest that eriocitrin is a powerful peppermint antioxidant and a free radical scavenger.

The plant phenolic compounds such as flavonoids, tannins and phenolic acids appeared to be strong antiradical and antioxidant compounds. The number of hydroxy groups and the presence of a 2,3-double bond and orthodiphenolic structure enhance antiradical and antioxidative activity of flavonoids. The glycosylation, blocking the 3-OH group in C-ring, lack of a hydroxy group or the presence of only a methoxy group in B-ring have a decreasing effect on antiradical or antioxidative activity of these compounds. Tannins show strong antioxidative properties. Some tannins in red wine or gallate esters were proved to have antioxidative effect in vivo. The number of hydroxy groups connected with the aromatic ring, in ortho or para position relative to each other, enhance antioxidative and antiradical activity of phenolic acids. The substitution of a methoxy group in ortho position to the OH in monophenols seems to favour the antioxidative activity of the former.

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source.

The aim of this work was to characterize the redox properties of the new antioxidant 5,8- dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochloë odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8- DHC at pH 7.0 was characterized by the midpoint potential (E p/2 ) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H 2 O 2 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP + reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 ± 5.5) μm. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N’-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-omethyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.

To validate the potential as added-value resources of Asteraceae and Labiatae species of Argentinean semi-arid lands, we have selected 13 of their major terpenoids belonging to several chemical classes and tested their insect antifeedant and toxic activity on the herbivorous insects Spodoptera littoralis and Leptinotarsa decemlineata. The antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedant to L. decemlineata was the eudesmane sesquiterpene γ-costic acid (13), followed by the labdane diterpene 2α,3α-dihydroxycativic acid (8), the clerodane diterpenes 6-acetylteucjaponin B (5), bacchotricuneatin A (1), bartemidiolide (7), butanolide (4), and the sesquiterpenes ilicic acid (11) and tessaric acid (10) (eudesmane and eremophilane type, respectively). S. littoralis was only affected by the clerodanes and showed the strongest response to salviarin (3) and 5, followed by hawtriwaic acid (6) and 12-epi-bacchotricuneatin A (2). Orally injected S. littoralis larvae were negatively affected by 5. Most of the diterpenes had selective cytotoxic effects to insect-derived Sf9 cells with the clerodane 1 being the most active, followed by the eudesmane costic acid (12), the only cytotoxic sesquiterpene. None of these compounds was cytotoxic to mammalian CHO cells.

A pathogenic isolate of Fusarium, F. oxysporum f. sp. batatas O-17 (PF), causes wilt disease in leaf etiolation in sweet potato (Ipomoea batatas) and morning glory (Ipomoea tricolor). Extracts from PF cultures were screened for phytotoxic components using a growth inhibition assay with morning glory seedlings. The extracts were fractionated using differential solvent extraction and two active compounds, ergosterol and fusalanipyrone, were isolated from the less-polar fraction. Growth inhibition of morning glory seedlings showed a sigmoidal doseresponse relationship, with fusalanipyrone exhibiting a two order of magnitude higher EC 50 value than ergosterol (18 nᴍ and 1.6 μᴍ, respectively). Both compounds showed lower growth inhibition activity towards lettuce seedlings (Lactuca sativa). This study provides information on the phytotoxic components of PF and discusses the mechanism behind PFfinduced phytotoxicity.

Hairy root cultures were established after inoculation of Genista tinctoria in vitro shoots with Agrobacterium rhizogenes, strain ATCC 15834. In transformed roots of G. tinctoria grown in Schenk-Hildebrandt medium without growth regulators the biosynthesis of isoflavones, derivatives of genistein and daidzein, and flavones, derivatives of luteolin and apigenin, characteristic for the intact plant, was completely inhibited. The only compound synthesized in G. tinctoria hairy roots was isoliquiritigenin (2.3 g/100 g DW), a daidzein precursor absent in the intact plant. This compound was stored entirely within cells and it was not until abscisic acid was added (37.8 μm supplement on day 42) that approx. 80% of it was released into the experimental medium. The paper discusses the effect of abscisic acid on the growth of G. tinctoria hairy root cultures, the biosynthesis of isoliquiritigenin and the way it is stored. A prototype basket-bubble bioreactor was designed and built to upgrade the scale of the G. tinctoria hairy root cultures. With immobilized roots and a new aeration system, large amounts of biomass were obtained (FW max 914.5 g l -1 ) which produced high contents of isoliquiritigenin (2.9 g/100 g DW). The abscisic acid-induced release of the metabolite from the tissue into the growth medium greatly facilitated subsequent extraction and purification of isoliquiritigenin.

The influence of electric field treatment on dedifferentiation and calli formation on rape hypocotyls was investigated. Segments, 10 mm long, of the upper part of rape (Brassica napus L., cv. GórczaΔski) hypocotyls were stimulated by different combinations of voltage/time (1.5 V/120 h, 3 V/3 h, 10 V/15 min and 30 V/30 s) under in vitro conditions. With all electric field treatments, segments oriented with their apical part towards the cathode produced more calli as compared to control (non-treated with electric field). Under opposite orientation slight inhibition of callus growth was observed. As the strongest effect on callus growth was observed after treatment with 30 V/30 s, this electric field treatment was selected for following analyses: the incorporation of [ 14 C]-2,4-D (2,4-dichlorophenoxyacetic acid) and [ 14 C]- BAP (benzylaminopurine) from the culture medium, changes in ACC (1-aminocyclopropane- 1-carboxylic acid) level and the redox activity in apical and bottom parts of hypocotyls during 18 d of culture. In contrast to changes in fresh weight, electric field treatment (30 V/30 s) stimulated a higher accumulation of 2,4-D and BAP in basal parts of hypocotyls than in apical ones. Moreover, orienting the apical part towards the cathode resulted in lower uptake of hormones as compared with the opposite orientation. The ACC concentration increased, especially in the basal parts of hypocotyls, independently on electric field application. However, the highest level was observed after electric field treatment with orientation of the apical part towards the anode. The distribution of oxidative substances (measured as the amount of ferric ions) between the apical and bottom part of hypocotyls was not changed when the apical parts were oriented towards the cathode. Under these conditions a decrease in apical and an increase in basal parts was observed during culture. Opposite orientation influenced the redistribution of oxidative substances from the first day of electric field treatment. Based on these results we suggest that electric field action can be connected with its influence on specific concentration of oxidative substances and hormone distribution in cells.

The metabolism of 14 C-4-n-nonylphenol ( 14 C-4-n-NP), as a mo-el for the xenoestrogen nonylphenol, was investigate- in three types of tobacco cell suspension cultures: one genetically non-modified culture (NT) and two cultures constitutively expressing human cytochrome P450 CYP1A1 or CYP1A2. With 1 mg l -1 of 14 C-4-n-NP and 24 h of incubation, the xenobiotic was transformed almost completely to glycosides. After glycosidic cleavage, 14 C-4-n-NP and several primary metabolites of 14 C-4-n-NP were liberated. Portions of the primary metabolites were 29.3% (NT culture), 34.3% (CYP1A1 culture), and 50.7% of applied 14 C(CYP1A2 culture). Thus, the endogenous capacity of the tobacco cells to form primary metabolites of 4-n-NP was noticeably higher than that of CYP1A1 or CYP1A2. The results however clearly suggest that 4-n-NP is - even though a poor - substrate of CYP1A1 an- CYP1A2. In order to examine metabolic profiles of 4-n-NP in the NT, CYP1A1 and CYP1A2 cultures, the suspensions were exposed to 10 mg l -1 of 14 C-4-n-NP using a two-liquid-phase system with carrier n-hexadecane and 192 h of incubation. Results obtained resembled those of the low concentration study. The oxidative metabolic profiles determined after hydrolytic cleavage using GC-EIMS were similar in the NT, CYP1A1 and CYP1A2 cultures. Main metabolites were side-chain mono-hydroxylated derivatives of 4-n-NP with 6′-, 7′- and 8′- OH-4-n-NP as prominent metabolites. In addition, olefinic side-chain hydroxy, ring methoxylated, keto and ring hydroxylated derivatives were observed. The lack of differences in metabolic profiles among the CYP1A1, CYP1A2 and NT cultures was referred to the low enzymatic activity of CYP1A1 and CYP1A2 as compared to the higher endogenous oxidative capacity of tobacco, as well as to similar metabolic profiles of 4-n-NP produced by CYP1A1 and CYP1A2 an- tobacco itself.

Nine strains of Erwinia amylovora were isolated from new host plants in Bulgaria D chokeberry and strawberry. The strains were characterized morphologically and biochemically using the API 20E and BIOLOG system. It was established that they showed three different API 20E metabolic profiles, not found by previous studies of E. amylovora. All strains were identified as E. amylovora due to their metabolic fingerprint patterns obtained by the BIOLOG system. The identification was confirmed by PCR amplification of a specific region of plasmid pEA29 and genome ams-region. This study is the first characterization and identification of E. amylovora strains isolated from chokeberry and strawberry by the API 20E and BIOLOG system and by polymerase chain reaction.

The presence of multiple forms of enzyme with terminal action pattern on pectate was evaluated in the protein mixture obtained from carrot roots. The form with pH optimum 3.8 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates). Its molecular mass, isoelectric point, glycosylation as well as cleavage of pectate from nonreducing end corresponded to an exopolygalacturonase [EC 3.2.2.67]. The affinity of this enzyme to the substrates increased with the increasing degree of polymerization, and the difference was observed only in the maximal ratio of catalysis of oligomeric and polymeric substrates. Sterical hindrance for substrates with more than six ᴅ-galactopyranuronic acid units is supposed and an oligogalacturonate hydrolase rather than exopolygalacturonase is considered.

We extracted silk produced by the larva of the hornet Vespa simillima xanthoptera Cameron from its nest. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the extracted hornet silk showed four major components with molecular weights between 35 and 60 kDa. The main amino acid components of the hornet silk protein were Ala (33.5%), Ser (16.9%), Asp (8.5%) and Glu (8.1%). The hornet silk could be dissolved in hexafluoroisopropyl alcohol (HFIP) at 25 °C without incurring molecular degradation. A transparent film of hornet silk was obtained readily by the formation of a cast upon drying of the hornet silk in the HFIP solution. Residual HFIP solvent was removed from the film by extraction with pure water. Solid-state 13 C NMR and FT-IR measurements revealed that the secondary structures of hornet silk proteins in the native state consisted of coexisting α-helix and β-sheet conformations. The β-sheet to α-helix ratio, which was changed by processing, was mainly responsible for the silk’s thermostability.

Although chemoreception plays an important role in social organization of many lizards, only a few studies have examined the chemicals found in secretions used for intraspecific communication. We report the composition of the secretion of the femoral glands of males of the spiny-footed lizard (Acanthodactylus erythrurus). On the basis of mass spectra, obtained by GC/MS, we identified 45 lipophilic compounds, including several alcohols ranging from 10 to 29 carbon atoms (mainly hexacosanol and tetracosanol), steroids (mainly cholesterol and dehydrocholesterol), n-C 9 to n-C 20 carboxylic acids, esters of carboxylic acids, and minor components such as lactones, ketones, squalene and α-tocopherol. Some of these compounds are reported for the first time in lizards. Adult and subadult males differed in the composition of secretions, with C 9 to C 15 carboxylic acids being more abundant in younger than in older lizards, whereas C 16 to C 20 carboxylic acids were more abundant in older lizards. Also, older lizards had significant lower proportions of cholesterol and campesterol but higher proportions of dehydrocholesterol.

Carboxymethylated derivatives were prepared from the (1→3)-β-ᴅ-glucan isolated from the cell wall of baker’s yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-α by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-α release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1→3)-β-ᴅ-glucan, are potent macrophage stimulators and activators of TNF-α release, which implies their potential application in antitumor therapy.

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with M r = (90.6 ± 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02 -1.0 mg/ml, presence of up to 100 mm phosphate and up to 200 mm NaCl, temperature in the range 4D25 °C. The sedimentation coefficient (6.04 ± 0.02) S, together with the diffusion coefficient (6.15 ± 0.11) 10 -7 cm 2 /s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mm Hepes, pH 7.0, yielded a molecular mass of (90.2 ± 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 μg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.

A variety of alkaloids, most of which occur or are structurally related to alkaloids that occur in skin glands of dendrobatid poison frogs, were assayed for antimicrobial activity against the Gram-positive bacterium Bacillus subtilis, the Gram-negative bacterium Escherichia coli and the fungus Candida albicans. Certain pyrrolidines, piperidines and decahydroquinolines, perhydro-histrionicotoxin, and a synthetic pumiliotoxin were active against B. subtilis. Only 2-n-nonylpiperidine was active against E. coli. One pyrrolidine, two piperidines, two decahydroquinolines, and the synthetic pumiliotoxin were active against the fungus C. albicans. The results suggest that certain of the skin alkaloids of poison frogs, in addition to being noxious to predators, may also benefit the frog through protection against skin infections.

Cancer patients’ responses to radiotherapy vary in severity. It has been suggested that it may be due to differences in intrinsic cellular radiosensitivity. Prediction of tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Towards this goal the micronucleus and apoptosis tests have been proposed as methods for measurement of chromosomal damage in peripheral blood lymphocytes. In this study, gamma-ray sensitivity of cultured lymphocytes of 26 breast cancer patients with early or late reactions was investigated. After irradiation with 4 Gy gamma radiation in G 0 , the frequency of micronuclei for patients with early reactions was significantly higher (P < 0.05) than for patients with late reactions. In the contrary the frequency of apoptosis for patients with early reactions was significantly lower (P < 0.05) than in the other group. It could be suggested that such a reduced amount of micronuclei in the late effects group is due to the presence of some residual DNA damages which are not completely repaired and lesions show increasing severity when the patients’ cells are irradiated again. These induced damages, probably are high enough to stimulate other endpoints like apoptosis instead of micronuclei.

Hydroxylamine and HPLC methods, measuring in vitro kinetics of enzymatic hydrolysis of acetylcholine or acetylthiocholine by cholinesterases, are described. The hydroxylamine method determines the dependence of substrate concentration vs. time, the HPLC method is able to measure simultaneously the time dependences of substrate and both primary products, choline or thiocholine, and acetic acid. Practical determinations are shown, comparison with known (above all Ellman’s and pH-stat) methods, advantages and disadvantages are discussed.