Volume 59, Issue 5-6

In the course of our investigations of new sources of higher plant lipids, seed fatty acid compositions and the tocochromanol contents of Salvia bracteata, S. euphratica var. euphratica, S. aucherii var. canascens, S. cryptantha, S. staminea, S. limbata, S. virgata, S. hypargeia, S. halophylla, S. syriaca and S. cilicica were investigated using GLC and HPLC systems. Some of the species are endemic to Turkey. All the Salvia sp. showed the same pattern of fatty acids. Linoleic, linolenic and oleic acid were found as the abundant components. Tocochromanol derivatives of the seed oil showed differences between Salvia species. γ-Tocopherol was the abundant component in most of the seed oils except of S. cilicica. The total tocopherol contents of the seed oils were determined to be more than the total of tocotrienols.

Three species of red marine macro algae (Rhodophyta) from the Indian Ocean were analysed for the occurrence of conjugated polyenes. The composition of different lipid classes in these seaweeds along with their fatty acid composition has also been reported. Analysis of lipid classes of these seaweeds revealed that both Acanthophora spicifera (Ceramiales, Rhodophyta) and two species of Gracilaria, viz. G. edulis and G. folifera (Gracilariales, Rhodophyta) were rich in glycolipids followed by neutral- and phospholipids. The fatty acid composition of these seaweeds revealed C16:0 as the predominant fatty acid in all three species. However, A. spicifera had significantly higher amounts of eicosapentaenoic acid (EPA) and arachidonic acid (AA) as compared to negligible amount of these fatty acids in both species of Gracilaria. The red seaweed Acanthophora spicifera contained conjugated eicosapentaenoic acid (CEPA) and conjugated arachidonic acid (CAA) in all lipid classes except glycolipids.

The resorcinolic lipid content and homologue composition of winter barley grains harvested at two field locations were evaluated. Depending on the crop location, the predominant alkylresorcinols identified were 1,3-dihydroxy-5-n-heneicosylbenzene or 1,3-dihydroxy- 5-n-pentacosylbenzene. Both resorcinol concentration and their homologue profiles were diverse in samples harvested at different fields indicating a prevailing role of the environment upon the alkylresorcinol biosynthesis in cereals.

A mixture of five saturated 5-n-alkylresorcinol homologues was isolated from vegetative cells of the nitrogen-fixing soil bacterium Azotobacter chroococcum Az12. Their structures were established by spectrometry ( 1 H NMR, EI-MS, FAB-MS, FAB-MS/MS) and chromatography (GC, TLC) means.

The oil of Adenanthera pavonina L. seeds was analysed by chromatographic and instrumental means. The oil was found to be rich in neutral lipids (86.2%), and low in polar lipids (13.8%). The neutral lipids consisted mainly of triacylglycerols (64.2%). Unsaturated fatty acids were found as high as 71%, while the percentage of saturated fatty acids was only 29%. GC and GC/MS analyses revealed linoleic, oleic and lignocerotic acid to be predominant among all fatty acids in the A. pavonina oil, whereas stigmasterol was the major steroid identified within this study. Subsequently, the oil was used for preparation of submicron oilin- water (o/w) lipid emulsions. Lipid emulsions were formulated by using soybean lecithin (SL) to investigate their particle size, Zeta potential and stability at the different oil and SL ratios. The results obtained indicate possible applications of the tested oil in pharmaceutical and medical fields as drug and cosmetic active ingredient carriers.

The essential oil of Elephantopus scaber, a famous medicinal herb from Yangjiang County in Southern China was analyzed for the first time using GC-MS. The major constituents were hexadecanoic acid (42.3%), isopropyl dimethyl tetrahydronaphthalenol (14.1%), β-sesquiphellandrene (8.3%), octadecadienoic acid (5.5%), and phytol (5.2%).

The seeds of the almond tree [(Prunus dulcis (Mill.) D. A. Webb. (syn. Prunus amygdalus)] were collected in two different periods of maturity and were studied for their lipid content. The total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and were analyzed by HPTLC coupled with a flame ionization detector (HPTLC/FID) and GC-MS. The oils were found to be rich in neutral lipids (89.9% and 96.3% of total lipids) and low in polar lipids (10.1% and 3.7% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of phospholipids. GC-MS data showed that the main fatty acid for both oils was 9-octadecenoic acid (oleic acid). The unsaturated fatty acids were found as high as 89.4% and 89.7%, while the percentage of the saturated fatty acids was found 10.6% and 10.3% for the immature and mature seed oils, respectively. Liposomes were prepared from the isolated phospholipids using the thin lipid film methodology, and their physical properties were characterized. Cytotoxicity was found absent when assayed against normal and cancerous cell lines. These new formulations may have future applications for encapsulation and delivery of drugs and cosmetically active ingredients.

Bioactivity-guided fractionation of Polygala alpestris L. (Rchb.) extracts led to the identification of two new xanthones, 1,3,7-trihydroxy-2,6-dimethoxyxanthone (1) and 2,3-methylenedioxy-4,7-dihydroxyxanthone (2). In addition five known compounds 3,4-dimethoxy-1,7-dihydroxyxanthone (3), 1,3-dihydroxy-7-methoxyxanthone (4), 1,7-dihydroxy-2,3-dimethoxyxanthone (5), 3′,6-O-disinapoyl sucrose (6) and 3′,5′-dimethoxybiphenyl-4-olo (7) were isolated. The structures of the isolated compounds were established by means of high resolution mass spectrometry, mono- and bi-dimensional NMR spectroscopy. All isolated compounds were tested for cytotoxic activity against three tumor cell lines (LoVo, HL-60, K 562).

Nine halogenated monoterpenes isolated from the red alga Plocamium cartilagineum have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the mammalian non-tumor cell line CHO (Chinese hamster ovary cells). The activities of these compounds were compared with those of the insecticide γ-hexachlorocyclohexane (lindane) due to chemical structure similarities. Compounds 1, 2, 3, and 5 exhibited selective cytotoxicity against colon and cervical adenocarcinoma cells. Interestingly, the effect of compound 3 was specific and irreversible to human colon adenocarcinoma SW480 cells, which overexpress the transmembrane P-glycoprotein often related to chemoresistance. None of the anti-tumor doses of these compounds was cytotoxic against CHO cells. Furthermore, analysis of cellular extracts after incubation with the test compounds and rotenone (positive uptake control) demonstrated the intracellular accumulation of 1, 2, 3, and 5.

Tagetes mendocina (Asteraceae) is a medicinal plant widely used in the Andean provinces of Argentina. Preliminary assays showed free radical scavenging activity in the methanol extract of the aerial parts, measured by the decoloration of a methanolic solution of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and scavenging of the superoxide anion. Assayguided isolation led to 4′-hydroxyacetophenone (1), protocatechuic acid (2), syringic acid (3), patuletin (4), quercetagetin 7-O-β-d-glucoside (5), patuletin 7-O-β-d-glucoside (6) and axillarin 7-O-β-d-glucoside (7) as the free radical scavengers and antioxidant compounds from Tagetes mendocina. On the basis of dry starting material, the total phenolic content of the crude drug was 3.00% with 0.372% of flavonoids. The content of compounds 1-7 in the crude drug was 0.008, 0.015, 0.010, 0.029, 0.238, 0.058 and 0.017%, respectively. Quercetagetin 7-O-β-d-glucoside proved to be the main free radical scavenger of the extracts measured by the DPPH decoloration test as well as for quenching the superoxide anion and inhibition of lipoperoxidation in erythrocytes. In the lipid peroxidation assay the percentual inhibition was related with the number of methoxy groups in the molecule, ranging from 86% for the quercetagetin glucoside to 67% for the monomethoxylated and 31% for the dimethoxylated derivative. The compounds showed low cytotoxicity towards human lung fibroblasts with IC 50 > 1mᴍ for compounds 1-3 and 0.24 to 0.52 mm for the flavonoids 4-7.

Some current oils and their main components were studied to determine their antioxidant values. This was done by using the modified method of ferric reducing ability of plasma. It has been established that volatile oils of medicinal plants have on average a reducing capacity of 3.5-220 mmol/kg oil. The reducing capacities of the main constituents of volatile oils are 0.165-65.5 mmol/kg in concentrated oils. The highest reducing capacity was showd for phellandrene (65.438 ± 0.166 mmol/kg) and anethole (50.087 ± 0.160 mmol/kg) while the lowest values were obtained for menthol (0.165 ± 0.023 mmol/kg) and menthone (0.168 ± 0.010 mmol/kg). It has been stated that the antioxidant values of the main constituents are lower than those of volatile oils. The reducing capacity of the main constituents of medicinal plant drugs at different concentrations was also determined.

The antioxidant activity in vitro of three poly(phenylacetyloxy)-substituted 1,1′:4′,1″-ter-phenyl compounds from the edible mushroom Thelephora ganbajun were investigated. The IC 50 values of compounds 1-3 for lipid peroxidation in rat liver homogenate were 400, 48, 54 μᴍ, respectively. Compounds 1-3 increased superoxide dismutase (SOD) activity with EC 50 values of 182, 74, 204 μᴍ. They were also assessed on the DPPH (1,1-diphenyl-2-picryl- hydrazyl) radical scavenging activity with EC 50 values of 49, 1233, 55 μᴍ.

A methanol extract was obtained from defatted (petroleum ether) inflorescence of Helichrysum arenarium (L.) Moench (perennial herb native to Middle and Southeast Europe). The extract was evaporated under reduced pressure and the dry residue was dissolved in hot water. The aqueous solution was stored for 6 d at 4 °C and the precipitate discarded. The remaining solution was divided into three aliquots a, b and c. Part a was extracted with ethyl acetate to obtain extract (A), part b was extracted with diethyl ether to obtain extract (B) and part c was subjected to alkaline hydrolysis and then extracted with diethyl ether to obtain extract (C). Extracts (A), (B) and (C) were evaporated under reduced pressure to obtain the dry residues A, B and C which were further investigated for phenolic compound content by TLC and HPLC and for antiradical activity with 2,2-diphenyl- 1-picrylhydrazyl radical (DPPH • ) as a substrate. Residue C exhibited stronger antiradical properties than non-hydrolysed residues A and B. HPLC analysis showed a great increase of caffeic acid in residue C. We concluded that the hydrolysis process led to a significant increase of free caffeic acid (strong antioxidant) concentration resulting in increased antiradical activity of residue C.

The chemical composition of the essential oils obtained from the aerial parts of Helichrysum cymosum and H. fulgidum, from Tanzania, were analyzed by GC and GC/MS. A total of sixty-five compounds, representing 92.4% and 88.2% of the two oils, respectively, were identified. trans-Caryophyllene, caryophyllene oxide, β-pinene, p-cymene, spathulenol and β- bourbonene were found to be the main components. Furthermore, the oils were tested against six gram (±) bacteria and three pathogenic fungi. It was found that the oil of H. fulgidum exhibited significant antimicrobial activity, while the oil of H. cymosum was not active at all.

The composition of the essential oil of the fruits, leaves and stems of Daucus glaber Forssk has been studied by GC/MS. It was found that, the essential oil of the fruits consists of monoterpene hydrocarbons (limonene and sylvestrene are the majors) and phenylpropanoids (elemicin is the major). Sylvestrene has never been reported before in the essential oil of any Daucus species. The study of the essential oil of the leaves revealed the presence of monoterpene hydrocarbons; limonene and γ-terpinene are the majors and a small amount of sylvestrene. The essential oil of stems consists of monoterpene hydrocarbons (γ-terpinene is the major), terpene alcohols (mainly 4-terpineol) and phenylpropanoids (myristicin and elemicin are the majors). It is interesting that, the essential oil of the fruits is free from any oxygenated terpenes while that of the stems is free from limonene and sylvestrene which are present in the essential oil of the fruits and leaves in fairly large amounts. The essential oil of the fruits, leaves and stems shows broad antimicrobial activities against both gram positive and gram negative bacteria. In addition, the volatile oil of the stem, particularly, show activi­ties against Candida albicans (yeast). Also, the prepared oils have variable cytotoxic activities with LC 50 21.52, 36.01 and 42.34 μrg/ml. respectively.

Tulasnein (1), a new metabolite with strong antimicrobial and weaker cytotoxic and phyto­toxic activity, was isolated from culture filtrates of three strains of the xylariaceous coprophilous fungus Podosordaria tulasnei. The producing strains were identified by their rhizomorphs and by ITS rDNA sequence analysis. A second new metabolite, podospirone (2), was also produced by all three strains whereas the weakly cytotoxic (+)-3,4-anhydroshikimic acid methyl ester (3) was detected in only one strain.

The acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid constituent showed antimicrobial activity against Bacillus subtilis, Listeria monocytogenes, Proteus vul­garis, Staphylococcus aureus, Streptococcus faecalis. Yersinia enterocolitica, Candida albicans, and Candida glabrata. Norstictic acid was active against Aeromonas hydrophila as well as the above microorganisms except Yersinia enterocolitica. Protocetraric acid showed activity only against the tested yeasts Candida albicans and Candida glabrata. The MIC values of the extract as well as of the three substances were determined. No antifungal activity of the acetone extract has been observed against ten filamentous fungi.

The cyclic monoterpene ketone (−)-carvone was metabolized by the plant pathogenic fungus Absidia glauca. After 4 days of incubation, the diol 10-hydroxy-(+)-neodihydrocarveol was formed. The absolute configuration and structure of the crystalline substance was identified by means of X-ray diffraction and by spectroscopic techniques (MS, IR and NMR). The antimicrobial activity of the substrate and metabolite was assayed with human pathogenic microorganisms.

We studied the effect of exogenous alkylresorcinols on the lipid metabolism of Azotobacter chroococcum. We observed that when 5-n-pentadecylresorcinol was present in the growth medium, the more endogenous alkylresorcinols were synthesized. Concurrently, a drop in the amount of phospholipids was observed. These changes were associated with increasing numbers of dormant cysts, while the number of vegetative cells diminished. The chemical nature of the alkylresorcinols synthesized by Azotobacter chroococcum was dependent on the duration of exposure of the bacteria to exogenous alkylresorcinols. When the exposure time was prolonged to four days, 5-n-nonadecylresorcinol (C 19:0) was substituted by 5-nheneicosylresorcinol (C 21:0) and 5-n-tricosylresorcinol (C 23:0). Two fluorescent membrane probes, NBD-PE and TMA-DPH, further revealed that the presence of alkylresorcinols in the lipid bilayer restrains the phospholipid rotational motion.

The toxicity of conventional nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) is caused by their enzymatic free radical formation with the subsequent oxidative stress, the formation of alkylating nitroso and/or hydroxylamino metabolites, and oxyhemoglobin oxida­tion into methemoglobin. In order to get an insight into the mechanisms of toxicity of the novel explosives NTO (5-nitro-1,2,4-triazol-3-one) and ANTA (5-nitro-1,2,4-triazol-3-amine), we examined their reactions with the single-electron transferring flavoenzymes NADPH: cytochrome P-450 reductase and ferredoxin:NADP + reductase, two-electron transferring fla­voenzymes mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase), and Enterobacter cloacae NAD(P)H:nitroreductase, and their reactions with oxyhemoglobin. The reac­tivity of NTO and ANTA in the above reactions was markedly lower than that of TNT. The toxicity of NTO and ANTA in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. This points to the involvement of oxidative stress in their cytotoxicity, presumably to the redox cycling of free radicals. The FLK cell line cytotoxicity and the methemoglobin formation in isolated human erythrocytes of NTO and ANTA were also markedly lower than those of TNT, and similar to those of nitrobenzene. Taken together, our data demonstrate that the low toxicity of nitrotriazole explosives may be attributed to their low electron-accepting properties.

The microbial transformation of coumarin (1) and 6-methyl coumarin (2) using Colletotrichum capsici gave 2-(3′-hydroxypropyl) phenol (3) and 2-(3′-hydroxypropyl)-4-methyl phe­nol (4). The phytopathogenic fungi effectively reduced the 2H-pyran-2-one moiety of both parent coumarins to respective alcohols.

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mᴍ) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mm NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.

In the course of our investigations of new sources of higher plant lipids, seed fatty acid compositions and the tocochromanol contents of Salvia bracteata, S. euphratica var. euphratica, S. aucherii var. canascens, S. cryptantha, S. staminea, S. limbata, S. virgata, S. hypargeia, S. halophylla, S. syriaca and S. cilicica were investigated using GLC and HPLC systems. Some of the species are endemic to Turkey. All the Salvia sp. showed the same pattern of fatty acids. Linoleic, linolenic and oleic acid were found as the abundant components. Tocochromanol derivatives of the seed oil showed differences between Salvia species. γ-Tocopherol was the abundant component in most of the seed oils except of S. cilicica. The total tocopherol contents of the seed oils were determined to be more than the total of tocotrienols.

Phenytoin (diphenylhydantoin) is an antiepileptic agent effective against all types of partial and tonic-clonic seizures. Phenytoin limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord neurons maintained in vitro. This effect is mediated by a slowing of the rate of recovery of voltage activated Na + channels from inactivation. For this reasons it was thought of interest to study the binding affinities of phenytoin with cell membranes and their perturbing effects upon membrane structures. The effects of phenytoin on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that phenytoin interacts with cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that phenytoin perturbed a class of lipids found in the outer moiety of cell membranes; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the erythrocyte membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the insertion of phenytoin in the outer monolayer of the red cell membrane. This is the first time that an effect of phenytoin on the red cell shape is described. However, the effects of the drug were observed at concentrations higher than those currently found in plasma when phenytoin is therapeutically administered.

Costus spicatus, used in Brazilian traditional medicine to expel kidney stones, contains steroidal saponins with different chemical characteristics. In spite of its popular utilization as potent diuretic, no scientific reports correlate this activity with the chemical constituents of the extract. Therefore, two steroidal saponins (3β,22a,25R)-26-(β-ᴅ-glucopyranosyloxy)-2-methoxyfurost-5-en-3-yl O-ᴅ-apio-β-ᴅ-furanosyl-(1→2)-O-[6-deoxy-α-ʟ-mannopyranosyl- (1→4)]-β-ᴅ-glucopyranoside (1) and (3β,22α,25R)-spirostan-3-yl O-ᴅ-apio-β-ᴅ-furanosyl- (1→2)-O-[6-deoxy-α-ʟ-mannopyranosyl-(1→4)]-β-ᴅ-glucopyranoside (la), were isolated from the rhizomes of this plant and their effects on the Na + -ATPase and (Na + +K + )-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that 1 and la inhibit specifically the Na + -ATPase activity.

The metabolic modifications of birch (Betula pubescens Ehrh.) leaf phenolics in the digestive tract of its major defoliator, larvae of the autumnal moth Epirrita autumnata, were studied. The main phenolic acids of birch, i.e. chlorogenic and p-coumaroylquinic acids, were isomerised in the alkaline digestive tract. Moreover, only 16 to 92% of the ingested amounts of chlorogenic acid were found in the faeces of individual larvae; the missing portion is possibly being used in the formation of reactive o-quinones. Water-soluble flavonoid glycosides were mostly excreted unaltered. In contrast, lipophilic flavonoid aglycones were not excreted as such, but as glycosides after being detoxified by E. autumnata via glycosylation. When the larvae were fed with leaf-painted acacetin and kaempferide, i.e. two naturally occurring birch leaf flavonoid aglycones, acacetin-7-O-glucoside and kaempferide-3-O-glucoside appeared in larval faeces as major metabolites. However, the efficiency of aglycone glycosylation varied, ranging from 17 to 33%, depending on the aglycone and its dietary level. There was also large variation in the efficiency of glycosylation − from 2 to 57% − among individual larvae. These results demonstrate a compound-specific metabolism of phenolic compounds, leading to different phenolic profiles in the insect gut compared to its leaf diet.

There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences. There are data that GGR rates depend on the chemical nature of the lesions in a manner that the lesions inflicting larger distortion on the DNA double helix are repaired at higher rate. It is not known whether the TCR repair rates depend on the type of lesions and in what way. To address this question human cells were transfected with pEGFP and pEYFP plasmids treated with UV light, cis-diamminedichloroplatinum(II) (cisplatin) and angelicin and 24 h later the restored fluorescence was measured and used to calculate the respective NER rates. In a parallel series of experiments the same plasmids were incubated in repair-competent protein extracts to determine GGR rates in the absence of transcription. From the two sets of data, the TCR rates were calculated. We found out that cisplatin, UV light and angelicin lesions were repaired by GGR with different efficiency, which corresponded to the degree of DNA helix distortion induced by these agents. On the other hand the three lesions were repaired by TCR at very similar rates which showed that TCR efficiency was not directly connected with the chemical nature of the lesions.

We have confirmed through an enlarged set of 728 species with 10,000 or more compiled codons, and a subset of 237 species with at least 50,000 compiled codons, that the mean values of a previously described index Φ [the mean value of the ratio between the relative (G, C) content of Class II and Class I codons, where G and C are guanine and cytosine] decrease monotonically across five large taxa, viz archaea, bacteria, eukaryotes (excluding metazoa), metazoa (excluding vertebrates) and vertebrates. It is proposed that these main taxa diverge successively from an ancestral progenome along lines which have persisted over long periods of time, leading to a primordial non-symmetrical phylogenetic tree. Further divergence, i.e. from eukaryotes to plants, fungi and protozoans, has followed symmetrical branching with approximately equal numbers of replacements and fixations. A statistical analysis of the Φ values of twelve distinct proteins, distributed over more than one thousand species belonging to the five main groups, was made to verify whether older taxa have older proteins. This supposition was confirmed for the first four taxa, but it was inconclusive for the last pair, metazoa/vertebrates.