Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae).

Cave animals live under highly constant ecological conditions and in permanent darkness, and many evolutionary adaptations of cave-dwellers have been triggered by their specific environment. A similar "cave effect" leading to pronounced chemical interactions under such conditions may be assumed, but the chemoecology of troglobionts is mostly unknown. We investigated the defensive chemistry of a largely cave-dwelling julid group, the controversial tribe "Typhloiulini", and we included some cave-dwelling and some endogean representatives. While chemical defense in juliform diplopods is known to be highly uniform, and mainly based on methyl- and methoxy-substituted benzoquinones, the defensive secretions of typhloiulines contained ethyl-benzoquinones and related compounds. Interestingly, ethyl-benzoquinones were found in some, but not all cave-dwelling typhloiulines, and some non-cave dwellers also contained these compounds. On the other hand, ethyl-benzoquinones were not detected in troglobiont nor in endogean typhloiuline outgroups. In order to explain the taxonomic pattern of ethyl-benzoquinone occurrence, and to unravel whether a cave-effect triggered ethyl-benzoquinone evolution, we classed the "Typhloiulini" investigated here within a phylogenetic framework of julid taxa, and traced the evolutionary history of ethyl-benzoquinones in typhloiulines in relation to cave-dwelling. The results indicated a cave-independent evolution of ethyl-substituted benzoquinones, indicating the absence of a "cave effect" on the secretions of troglobiont Typhloiulini. Ethyl-benzoquinones probably evolved early in an epi- or endogean ancestor of a clade including several, but not all Typhloiulus (basically comprising a taxonomic entity known as "Typhloiulus sensu stricto") and Serboiulus. Ethyl-benzoquinones are proposed as novel and valuable chemical characters for julid systematics.

'Does my Diet Affect my Perfume?' Identification and Quantification of Cuticular Compounds in Five Drosophila melanogaster Strains Maintained over 300 Generations on Different Diets.

Cuticular hydrocarbons (CHCs) in Drosophila melanogaster represent the basis of chemical communication being involved in many important biological functions. The aim of this study was to characterize chemical composition and variation of cuticular profiles in five D. melanogaster strains. These strains were reared for approximately 300 generations on five diets: standard cornmeal medium and substrates prepared with apple, banana, tomato, and carrot. Differences in quantity and/or quality in CHCs were assumed as a result of activation of different metabolic pathways involved in food digestion and adaptations to the particular diet type. In total, independently of sex and strain, 66 chemical compounds were identified. In females of all strains, 60 compounds were identified, while, in males, 47 compounds were extracted. Certain new chemical compounds for D. melanogaster were found. MANOVA confirmed that CHC amounts significantly depend on sex and substrates, as well as on their interactions. Discriminant analysis revealed that flies belonging to 'apple' and 'carrot' strains exhibited the most noticeable differences in CHC repertoires. A non-hydrocarbon pheromone, cis-vaccenyl acetate (cVA) also contributed to the variation in the pheromone bouquet among the strains. Variability detected in CHCs and cVA may be used in the explanation of differences in mating behaviour previously determined in analyzed fly strains.

Antioxidative activity of diarylheptanoids from the bark of black alder (Alnus glutinosa) and their interaction with anticancer drugs.

Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-ß-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-ß-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI-H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.

Isolation, characterization, and in vitro cytotoxicity of new sesquiterpenoids from Achillea clavennae.

Further phytochemical investigation of the aerial parts of Achillea clavennae has resulted in the isolation of three new sesquiterpene lactones: two highly oxygenated germacranolides (1, 2) and the iso-seco-guaianolide 9(R)-acetoxy-3-O-methyl-iso-seco-tanapartholide (3). Eight known compounds were also found, of which 9α-acetoxycanin (5), sintenin (6), and oleanolic acid (7) were detected for the first time. The structures of the isolated compounds were elucidated by combined spectroscopic methods (1D and 2D NMR, HRESIMS, CIMS, and FTIR). While the predominant metabolite germacranolide sintenin (6) was not cytotoxic, the new iso-seco-guaianolide (3) displayed cytotoxicity comparable to that of cisplatin and the lactone apressin (4), inducing partly apoptotic death in human U251 and rat C6 glioma cell lines.

Further amphoricarpolides from the surface extracts of Amphoricarpos complex from Montenegro.

Analysis of composition of sesquiterpene lactone fraction of leaf cuticular neutral lipids of Amphoricarpos complex from two different localities in north Montenegro, i.e., canyon of river Tara (A. autariatus ssp. autariatus) and mountain Zeletin (A. autariatus ssp. bertisceus) afforded sesquiterpene lactones with guaianolide skeletons (17 compounds), so called amphoricarpolides, typical for this genus. Nine of them, 9-17, were new compounds, and their structures were elucidated by detailed analyses of IR, NMR, and MS data.

Diarylheptanoids from green alder bark and their potential for DNA protection.

Nine diarylheptanoids, 1-9, catechin (11), and a phenolic glucoside, 10, were isolated from the bark of green alder (Alnus viridis). Four of the isolated compounds, i.e., 2, 5, 8, 10, are new. The structures of 1-11 were determined on the basis of spectroscopic data. All isolated compounds were evaluated for their in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using cytokinesis-block micronucleus (CBMN) assay. Almost all of them exerted a pronounced effect of decreasing DNA damage of human lymphocytes, acting stronger than the known synthetic protector amifostine.

Chemical defense in millipedes (Myriapoda, Diplopoda): do representatives of the family Blaniulidae belong to the 'quinone' clade?

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, were characterized by GC-FID and GC/MS analyses, which showed the presence of a complex mixture of benzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The major compound in the secretions of both analyzed species was 2-methyl-1,4-benzoquinone (toluquinone). The second major constituent in the N. kochii secretion was 2-methyl-3,4-(methylenedioxy)phenol, while in that of C. phlepsii, it was 2-methoxy-3-methyl-1,4-benzoquinone. The defensive secretion of N. kochii also showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyl oleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemical composition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidae in the 'quinone' millipede clade.

Chemistry of the sternal gland secretion of the Mediterranean centipede Himantarium gabrielis (Linnaeus, 1767) (Chilopoda: Geophilomorpha: Himantariidae).

The geophilomorph centipede, Himantarium gabrielis, when disturbed, discharges a viscous and proteinaceous secretion from the sternal glands. This exudate was found by gas chromatography-mass spectrometry, liquid chromatography-high resolution mass spectrometry, liquid chromatography-mass spectrometry-mass spectrometry and NMR analyses to be composed of hydrogen cyanide, benzaldehyde, benzoyl nitrile, benzyl nitrile, mandelonitrile, mandelonitrile benzoate, 3,7,6O-trimethylguanine (himantarine), farnesyl 2,3-dihydrofarnesoate and farnesyl farnesoate. This is the first report on the presence of benzyl nitrile and mandelonitrile benzoate in secreted substances from centipedes. Farnesyl 2,3-dihydrofarnesoate is a new compound, while himantarine and farnesyl farnesoate were not known as natural products. A post-secretion release of hydrogen cyanide by reaction of mandelonitrile and benzoyl nitrile was observed by NMR, and hydrogen cyanide signals were completely assigned. In addition, a protein component of the secretion was analysed by electrophoresis which revealed the presence of a major 55 kDa protein. Analyses of the defensive exudates of other geophilomorph families should produce further chemical surprises.

Diarylheptanoids from Alnus glutinosa bark and their chemoprotective effect on human lymphocytes DNA.

A study of secondary metabolites from the bark of Alnus glutinosa led to the isolation of fourteen diarylheptanoids: oregonin (1), platyphylloside (2), rubranoside A (3), rubranoside B (4), hirsutanonol (5), hirsutenone (6), hirsutanonol-5-O-ß-D-glucopyranoside (7), platyphyllonol-5-O-ß-D-xylopyranoside (8), aceroside VII (9), alnuside A (10), alnuside B (11), 1,7-bis-(3,4-dihydoxyphenyl)-5-hydroxy-heptane-3-O-ß-D-xylopyranoside (12), (5S)-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-5-O-ß-D-glucopyranosyl-heptan-3-one (13), and (5S)-1,7-bis-(3,4-dihydroxyphenyl)-5-O-ß-D-[6-(3,4-dimethoxycinnamoylglucopyranosyl)]-heptan-3-one (14). All of the diarylheptanoids, except 1 and 5, were found in A. glutinosa for the first time, while 13 and 14 were new compounds. The structures were determined by spectroscopic techniques: 1D and 2D NMR, HR-ESI-MS, FTIR, UV, and CD. All isolated compounds were analyzed for an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using the cytokinesis-block micronucleus assay. The majority of them, including the new compounds 13 and 14, exerted a pronounced effect in decreasing DNA damage in human lymphocytes. Diarylheptanoids 1, 2, 5, 13, and 14 at a concentration of 1 µg/mL decreased the frequency of micronuclei by 52.8 %, 43.8 %, 63.6 %, 44.4 %, and 56.0 %, respectively, exerting a much stronger effect than the synthetic protector amifostine (17.2 %, c = 1 µg/mL).

A new depsidone of Lobaria pulmonaria with acetylcholinesterase inhibition activity.

The phytochemical investigation conducted on a foliose lichen, Lobaria pulmonaria(L.) Hoffm. (Lobariaceae), led to the isolation of a new depsidone in the form of its diacetate derivative which showed a moderate acetylcholinesterase inhibition activity (1 µg) in vitro. This is the first record of identified depsidone structure in searching for these inhibitors.

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

BACKGROUND: The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Cernjavski & Soska (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). METHODS: The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. RESULTS: The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. CONCLUSION: Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

Pyrrolizidine alkaloids and fatty acids from the endemic plant species Rindera umbellata and the effect of lindelofine-N-oxide on tubulin polymerization.

The examination of the aerial parts, roots, and seeds of the endemic plant Rindera umbellata is reported in this paper for the first time. Phytochemical investigation of R. umbellata led to the isolation and characterization of ten pyrrolizidine alkaloids and eleven fatty acids in the form of triglycerides. Pyrrolizidine alkaloids 1-9 were found in the aerial parts, 7 and 8 in the roots, and 6-10, together with eleven fatty acids, in the seeds of this plant species. The structures of compounds 1-10 were established based on spectroscopic studies (¹H- and ¹³C-NMR, 2D NMR, IR and CI-MS). After trans-esterification, methyl esters of the fatty acids were analyzed using GC-MS. The effect of lindelofine-N-oxide (7) on tubulin polymerization was determined.

Isolation and biological evaluation of jatrophane diterpenoids from Euphorbia dendroides.

From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1-6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most effective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multi-drug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.

A new prenylated flavanonol from Seseli annuum roots showing protective effect on human lymphocytes DNA.

A new prenylated flavanonol named seselinonol (1) was isolated from the roots of Seseli annuum, together with the well-known biologically active polyacetylenes falcarinol (2) and falcarindiol (3), and the prenylated furanocoumarin phellopterin (4). Its structure was elucidated by extensive spectroscopic analysis, including HR-ESI-MS, 1D- and 2D-NMR. Seselinonol and phellopterin were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The new compound exerted a beneficial effect by decreasing DNA damage of human lymphocytes.

Chemical composition of essential oils of Thymus and Mentha species and their antifungal activities.

The potential antifungal effects of Thymus vulgaris L., Thymus tosevii L., Mentha spicata L., and Mentha piperita L. (Labiatae) essential oils and their components against 17 micromycetal food poisoning, plant, animal and human pathogens are presented. The essential oils were obtained by hydrodestillation of dried plant material. Their composition was determined by GC-MS. Identification of individual constituents was made by comparison with analytical standards, and by computer matching mass spectral data with those of the Wiley/NBS Library of Mass Spectra. MIC's and MFC's of the oils and their components were determined by dilution assays. Thymol (48.9%) and p-cymene (19.0%) were the main components of T. vulgaris, while carvacrol (12.8%), a-terpinyl acetate (12.3%), cis-myrtanol (11.2%) and thymol (10.4%) were dominant in T. tosevii. Both Thymus species showed very strong antifungal activities. In M. piperita oil menthol (37.4%), menthyl acetate (17.4%) and menthone (12.7%) were the main components, whereas those of M. spicata oil were carvone (69.5%) and menthone (21.9%). Mentha sp. showed strong antifungal activities, however lower than Thymus sp. The commercial fungicide, bifonazole, used as a control, had much lower antifungal activity than the oils and components investigated. It is concluded that essential oils of Thymus and Mentha species possess great antifungal potential and could be used as natural preservatives and fungicides.

New aurone epoxide and auronolignan from the heartwood of Cotinus coggygria Scop.

New aurone epoxide, 2,10-oxy-10-methoxysulfuretin (14), and new auronolignan (15), named cotinignan A, were isolated by silica gel column and semipreparative HPLC chromatography from the methylene chloride/methanol extract of Cotinus coggygria Scop. heartwood. In addition, thirteen known secondary metabolites namely sulfuretin, 2,3-trans-fustin, fisetin, butin, butein, taxifolin, eriodictyol, 3',5,5',7-tetrahydroxyflavanone, 3',4',7-trihydroxyflavone, 3-O-methyl-2,3-trans-fustin, 3-O-galloyl-2,3-trans-fustin, ß-resorcylic acid and 3-O-ß-sitosterol glucoside were isolated as well. Their structures were elucidated by 1D and 2D NMR, HR-ESI-MS, IR and UV. Ten out of eleven isolated flavonoids possess 7, 3' and 4' hydroxy groups. These structural features could be considered as chemotaxonomic characteristic of flavonoids from C. coggygria. Cotinignan A (15) represents new subclass of secondary metabolites - auronolignans.

Antiglioma action of xanthones from Gentiana kochiana: Mechanistic and structure-activity requirements.

The present study identifies xanthones gentiakochianin and gentiacaulein as the active principles responsible for the in vitro antiglioma action of ether and methanolic extracts of the plant Gentiana kochiana. Gentiakochianin and gentiacaulein induced cell cycle arrest in G(2)/M and G(0)/G(1) phases, respectively, in both C6 rat glioma and U251 human glioma cell lines. The more efficient antiproliferative action of gentiakochianin was associated with its ability to induce microtubule stabilization in a cell-free assay. Both the xanthones reduced mitochondrial membrane potential and increased the production of reactive oxygen species in glioma cells, but only the effects of gentiakochianin were pronounced enough to cause caspase activation and subsequent apoptotic cell death. The assessment of structure-activity relationship in a series of structurally related xanthones from G. kochiana and Gentianella austriaca revealed dihydroxylation at positions 7, 8 of the xanthonic nucleus as the key structural feature responsible for the ability of gentiakochianin to induce microtubule-associated G(2)/M cell block and apoptotic cell death in glioma cells.

Stereochemistry and conformations of natural 1,2-epoxy-guaianolides based on 1D and 2D NMR data and semiempirical calculations.

From detailed study of 1D and 2D NMR spectra of ten natural 1,2-epoxyguaianolides (bis-1,2:3,4-epoxyguaianolides and guaianolide-1,2-epoxychlorohydrins), we identified general spectral traits helpful for stereochemical assignment of such sesquiterpene lactones. We found that the chemical shifts of certain (1)H and (13)C nuclei are consistently dependent on the configuration of 1,2-epoxy-ring which could be used as a simple rule for establishing this configuration. Then, from 1D and 2D (COSY, NOESY, HMQC, HMBC) NMR data, applying the observed rule, the structure and stereochemistry of two new, diastereomeric guaianolide-1,2-epoxychlorohydrins, isolated from Achillea serbica, are determined. The NMR data, namely, nuclear overhauser enhancement (NOE) correlations, pointed out two conformations of guaianolide's cycloheptane ring. The semiempirical calculations (AM1 and PM3 methods), performed in order to gain additional information regarding conformations, resulted in three geometries of investigated lactones. Even so, the conformations derived from the NMR data agreed well with those calculated by semiempirical methods.

Antioxidant activity of nine Fabaceae species growing in Serbia and Montenegro.

The aim of this study was to investigate antioxidant capacity of nine Fabaceae species collected on the mountains of Serbia and Montenegro. Antioxidant assays with various reaction mechanisms were used, including total phenolic content by Folin-Ciocalteu, DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) values by ABTS radical cation and inhibition of liposome peroxidation. The investigated plants exhibited strong antioxidant capacity in all the tested methods, and among them, Lathyrus binatus, Trifolium pannonicum, and Anthyllis aurea were found to be the most active.

Composition and antifungal activity of the essential oil of Seseli annuum wild-growing in Serbia.

The essential oil from the aerial parts of Seseli annuum, wild-growing in Serbia, was obtained by hydrodistillation and analyzed using GC and GC/MS. A total of 43 components were identified representing 96.5% of S. annuum oil. The most abundant compounds were germacrene D (29.8%), sabinene (10.3%), beta-ocimene Z (9.8%) and limonene (8.6%). The essential oil showed antifungal activity against fifteen fungi with MICs between 12.5 to 50 microl/ml.