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The isolation and identification of bioactive compounds from natural sources has given insights in the discovery of new drugs against several diseases. Labdane-type diterpenes isolated from nature, have been investigated in the past for their pharmaceutical activities. Labd-7,13-dien-15-ol (1), a major lipophilic constituent of the resin 'ladano' was found to exhibit cytotoxic and cytostatic activity against cell lines derived from solid tumors with a profound activity on small lung cancer cells, DMS114. The present study, based on liposomal technology, aimed to develop a suitable carrier of compound 1 to overcome its water insolubility that inhibits further in vivo administration. Liposomes have been proved adequate drug carriers which enhance the solubility of water insoluble drugs and reduce possible side effects. Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (EPC) and dipalmitoyl phosphatidylglycerol (DPPG) were used to prepare liposomes incorporating compound 1 at various molar ratios. Their physicochemical characteristics were determined and liposomes composed of EPC:DPPG 9:0.1 (molar ratio) was chosen as the most suitable carrier concerning their physical characteristics, stability, incorporation efficiency as well as the ability to retain the capture of compound 1 in the presence of RPMI-1640 medium. The activity of the liposomal formulation of compound 1 against human cancer cell lines was compared to that of its free form. The results showed that EPC:DPPG:1 9:0.1:5 (molar ratio) liposomal formulation maintained the pharmacological activity of 1, improved its water solubility and was justified as suitable formulation for in vivo administration of compound 1. © 2006 Elsevier SAS. All rights reserved.

Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidytcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations UP to X-sclareol = 9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (DeltaH) of the act to liquid-crystalline phase transition of DPPC bilayers. At concentrations X greater than or equal to 16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers; containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

The aim of this study was to synthesize simple thiol-reactive conjugates from maleimide and lipoamines (stearylamine or oleylamine) and to develop a simple, fast and low-cost method for the preparation of lyophilized general-purpose thiol-reactive liposomes. A formulation of egg phosphatidylcholine-dipalmitoylphoshatidylglycerol (9:0.1 molar ratio) was developed and characterized. Freeze-drying methodology was established to produce a stock of liposomes and the physicochemical characteristics of the reconstituted liposomes were compared with those of the initial preparation. The physicochemical properties (size and zeta-potential) of the new liposomal formulations were studied. High-performance thin-layer chromatography coupled to a flame ionization detector was applied for one-step analysis of the liposomal components and for determining the maleimide-lipoamine conjugates phospholipid molar ratio. The differences concerning the incorporation efficiency of the synthetic conjugates into liposomes were discussed on the basis of their conformational properties. The small difference in structure between the two thiol-reactive conjugates (i.e., the C18 alkyl chain double bond) causes a considerable difference in phospholipids packing of the resulting lipidic bilayers of the liposomes; the conformational bending of conjugate maleimide-oleylamine may contribute to the final architecture of liposomes.

Labd-13(E)-ene-8α,15-diol (1) and its derivative labd-13(E)-ene- 8α-ol-15-yl-acetate (2) are water insoluble biological active molecules and their structures were elucidated using NMR and X-ray techniques. Differential scanning calorimetry (DSC) was applied to study the thermal effects of 1 and 2 on DPPC bilayers. Liposomes composed of egg phosphatidylcholine/ dipalmytoylphosphatidylglycerol (9:0.1 molar ratio) were prepared by the thin-film hydration method and were used for incorporating 1 and 2. Free and liposomal 1 and 2 were tested for their activity against human cancer cell lines using the sulphorhodamine B assay. The effect of 1 and 2 on DPPC bilayers caused abolition of the pre-transition temperature, lowering of the main phase transition and reduction of the transition enthalpy only in the presence of cholesterol. The liposomes that have been designed and developed offer high incorporation efficiency; 62.4% (0.369 drug/lipid molar ratio) and 99.7% (0.661 drug/lipid molar ratio) for 1 and 2, respectively. Liposomal 2 showed growth-inhibiting activity against the majority of the tested cell lines. © 2005 Elsevier Ireland Ltd. All rights reserved.

Ten sesquiterpene lactones, 8α-O-(3,4-dihydroxy-2- methylenebutanoyloxy)sonchucarpolide (4-epi-malacitanolide) (1), 8α-O-(4-acetoxy-2-hydroxymethylbuten-2-oyloxy)-4-epi-sonchucarpolide (2), malacitanolide (3), its 4′-acetyl derivative (4), 8α-O-(3,4- dihydroxy-2-metnylenebutanoyloxy)dehydromelitensine (5), 8α-O-(3,4- dihydroxy-2-methylenebutanoyloxy)-15-oxo-5,7αH,6βH-eleman-1,3,11(13) -trien-6,12-olide (6), the germacranolides 8α-O-(4-acetoxy-2- hydroxymethylbuten-2-oyloxy)salonitenolide (7), cnicin (8), and 4′-acetylcnicin (9), and the sesquiterpene methyl 8α-O-(3,4- dihydroxy-2-methylenebutanoyloxy)-6α,15-dihydroxyelema-1,3,11(13) -trien-12-oate (10), were isolated from the aerial parts of Centaurea spinosa. Nine known flavonoids were also isolated. The structures and the stereochemistry of the new compounds 1 and 2 were deduced by spectroscopic methods. The in vitro activity of 1-10 against three Gram-positive and three Gram-negative bacteria was evaluated using a microdilution method, and their in vitro cytotoxic activity was determined against a panel of human tumor cell lines. © 2005 American Chemical Society and American Society of Pharmacognosy.

Liposomes composed of hexadecylphosphocholine/egg phosphatidylcholine/ stearylamine (HePC/EPC/SA) 10:10:0.1, 10:10:0.5 and 10:10:1 (molar ratio) (1-3) were prepared and lyophilized. The liposomes were physicochemically characterized (size and ζ-potential) and they were found stable at 4°C over a period of 4 weeks. In vitro, liposomes 1 and 2 were about twice more active than HePC against Leishmania donovani WT whereas liposomes 3 were about three times more active than HePC on HePC-resistant promastigotes. Although liposomes 1-3 were inactive on the in vitro intramacrophage amastigote model, the ability of the liposomes to accumulate within the liver where parasites are located justifies a further in vivo evaluation. We observed that liposome 1 was twice more active than HePC against Trypanosoma brucei brucei bloodstream forms maintained in vitro. In vivo results showed that liposomal HePC seemed to be less toxic than the free drug despite the absence of significant antitrypanosomal activity. © 2005 Elsevier SAS. All rights reserved.

Liposomes composed of HePC:EPC:SA 10:10:0.1 (molar ratio) (1) and EPC:SA 10:0.1 (molar ratio) (2) were prepared and were used for incorporating the doxorubicin-PAMAM complex (3:1 molar ratio) (3). The doxorubicin-PAMAM complex was attached to liposomes and the incorporation efficiency was 91 and 95% for 1 and 2, respectively. The incorporation efficiency for doxorubicin into PAMAM was almost 97% while doxorubicin to PAMAM molar ratio was 3.56 ± 0.04. The release rate of doxorubicin as doxorubicin-PAMAM complex from liposomes 1 and 2 and from the complex 3, was studied using buffers and 50% RPMI cell culture medium at 37 and 25°C. The low release rate of doxorubicin as well as the high incorporation efficiency of doxorubicin-PAMAM complex into liposomes are considered as beneficial factors concerning the activity of doxorubicin. The cytotoxic activity of the liposomal formulation 1 incorporating doxorubicin-PAMAM complex, based on doxorubicin activity, was compared to that of 2 incorporating doxorubicin-PAMAM complex and to that of 3. The results showed that complex 1 was the most active formulation against all cancer cell lines compared to that of 2 and 3. Liposomal formulations composed of lipids and of a drug-dendrimer complex could be characterized as modulatory liposomal controlled release system (MLCRS), and could provide benefits to the delivery of drugs and modulate their release. © 2005 Elsevier B.V. All rights reserved.

The chemical composition of the essential oils obtained from leaves and bracts of hydroponically cultivated Origanum dictamnus L. (Cretan dittany), growing under various electrical conductivity (EC) levels (2.0, 4.0, and 6.0 mS/cm), was studied, using the nutrient film technique (NFT). The analysis of the essential oil content was achieved by GC-MS technique, and totals of 41 and 38 different compounds were identified in both cases of large-leaved and narrow-leaved samples of leaves and bracts, respectively. Differences in the composition content and of the percentage of each of the constituents in the two studied samples (i.e., large-leaved and narrow-leaved) and within the essential oils of leaves and bracts in both samples were observed. Carvacrol and p-cymene were identified as the main constituents in all essential oils, whereas thymoquinone was found in higher percentage in the essential oils of large-leaved than in narrow-leaved plants. The results obtained from GC-MS analysis were submitted to chemometric analysis, and a phenotypic similarity of the essential oils of narrow-leaved O. dictamnus was observed, whereas the essential oils of large-leaved O. dictamnus showed two separate subgroups © 2005 American Chemical Society.

Liposomes were prepared from the isolated phospholipids of mature broad bean [Vicia faba L. (syn. Fabae calabaricae)] oil and their physical properties were studied. The method of preparation was the hydration of the thin lipid film, while the probe sonication methodology was used for reducing the size of the vesicles. The seeds of the broad bean were collected in two different periods of maturity and extracted by the Bligh-Dyer method, and the lipid classes were studied by HPTLC/FID. The oils were found to be rich in polar lipids (63.1% and 60.2% of total lipids) and low in neutral lipids (36.9% and 39.8% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides (34.2% and 32.3%) whereas the polar lipids mainly consisted of phospholipids (60.2% and 54.2%) for the mature and immature seed oils, respectively. Sphingolipids (8.9%) were identified only in the immature seed oil. The overall goal of this study was the preparation of a new liposomal formulation with physicochemical properties such as unique lipid composition, size and ζ-potential, which are important factors influencing drug delivery to the target tissues. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

lt has been shown that the partitioning of vinblastine in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) single and multiple bilayer dispersions induces partial interdigitation of the lipid alkyl chains. Similar behavior has been observed for abietic and ursodeoxycholic acids and may well be generalized for the partitioning of bulky amphoteric molecules, which tend to localize in the vicinity of the polar heads. For the present study, differential scanning calorimetry (DSC) has been employed to investigate the role of lipid molecular characteristics such as the alkyl chain length and the polarity of the head-group, as well as the impact of cholesterol upon vinblastine-induced interdigitation. It is found that vinblastine does not induce interdigitation in lipids with either shorter or longer alkyl chains than DPPC, or having head-groups of different polarity. In addition, it is shown that the presence of cholesterol in the lipid bilayer tends to modulate the phase behavior of the lipid/vinblastine bilayer system. Preliminary studies show that such properties directly affect the encapsulation efficiency and the pharmacokinetics of liposomes. (C) 2003 Elsevier B.V. All rights reserved.

Polyunsaturated fatty acids (PUFAs) have been shown to possess a considerable anti-tumor and anti-bacterial effect in vitro. In an attempt to achieve serum concentrations of these acids similar to those applied in vitro, a solution of ethyl ester of arachidonic acid (AA) was administered intravenously at 25 mg/kg within 10 min in six male rabbits. Blood samples were collected before and 60 min after infusion from catheters inserted in the hepatic veins and in the carotid artery. Analysis of serum fatty acids was performed by gas chromatography mass spectrometry. Elevated concentrations of elongated fatty acids were detected in the hepatic veins after infusion. Mean concentrations of arachidonate in the hepatic veins and the carotid arteries after infusion of AA were 2.77 and 3.73 muM, respectively. It is concluded that the intravenous administration of a solution of AA might result in increased hepatic biosynthesis of serum saturated and unsaturated fatty acids of elongated carbon chains. The increasing interest for the application of PUFAs in therapeutics renders further study mandatory to clarify the significance of these findings. (C) 2003 Elsevier Ltd. All rights reserved.

One promising class of antioxidant compounds is polyphenols, contained abundantly in pine bark extract (Pycnogenol®-pine bark extract). This medicinal extract is utilized for its anti-inflammatory properties. Its pharmacological action in skin depends on the kinetics of its absorption. In this study the dermal bioavailability of pine bark extract was investigated. Viable human skin, adapted on continuously perfused Franz cells, was applied with 5% (w/v) pine bark solution. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, and 12-hour intervals and analyzed for detection of pine bark extract constituents by high performance liquid chromatography (HPLC) (reversed phase column, isocratic conditions) coupled with an electrochemical detector (EC). Several constituents of pine bark extract such as gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, vanillin, and one unidentified constituent were detected. These findings indicate that pine bark extract is readily absorbed by human skin and can be used for topical application.

In an attempt to identify the structure of a sesquiterpene from Cistus creticus ssp. creticus proposed in the literature as 1,1,4a,6-tetramethyl-5- methylene-1,2,3,4,4α,5,8,8α-octahydronaphthalene, the synthesis of its cis isomer 2 was carried out in 11 steps and 9.5% yield. Comparison of the spectra of 2 and those reported earlier for the synthetic irons isomer 1 with the spectral profile of the isolated natural product indicated that the latter was not compatible with either 1 or 2. The correct structure was assigned, by detailed spectroscopic analysis of the natural product, as 6-isopropenyl-4,4a- dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene (3).

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.

The volatile composition of eight Stachys species has been studied. The investigated taxa were St. alopecuros (L.) Bentham., St. scardica (Griseb.) Hayek, St. cretica L. ssp. cretica, St. germanica L. ssp. heldreichii (Boiss.) Hayek, St. recta L., St. spinulosa L., St. euboica Rech. and St. menthifolia Vis., growing wild in Greece. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC-MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. Sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa. Furthermore, the obtained essential oils were tested against the following six bacteria: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), Bacillus subtilis (ATCC 10907), Bacillus cereus (clinical isolates), Micrococcus flavus (ATCC 10240), Staphylococcus epidermidis (ATCC 2228), as well as against the following five fungi: Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Epidermophyton floccosum (clinical isolates), Candida albicans (clinical isolates) and Trichophyton mentagrophytes (clinical isolates). The tested essential oils showed better activity against bacterial species than against fungi. Pseudomonas aeruginosa was the most resistant strain, as none of the essential oils was active against this strain. The essential oil of St. scardica has been proven most active against both bacteria and fungi. © 2003 Elsevier Ltd. All rights reserved.

Essential oils from hydroponically cultivated Salvia fruticosa were analyzed by GC-MS techniques. Three different levels of nitrogen (100, 150, and 200 mg/L) were used in the nutrient solution for the cultivation, using the nutrient film technique. A total of 79 compounds were identified, and qualitative and quantitative differences have been observed between the samples collected at full bloom (flowering stage) and at the end of the seed formation stage. 1,8-Cineole, β-caryophyllene, and viridiflorol were the predominant constituents in most cases. 13-epi-Manool was identified by using GC parameters and mass spectrum fragmentation pattern, whereas labd-7,13-dien-15-ol, a labdane type diterpene, was identified for the first time in the genus Salvia, using GC parameters and an authentic sample. The results obtained from GC-MS analyses were submitted to chemometric analysis.

Sesquiterpene lactones isolated from the aerial parts of various species of the genus Centaurea were examined for their in vitro cytotoxic/cytostatic activity against five human cell lines (i.e., DLD1, SF268, MCF7, H460 and OVCAR3). Compounds 1-4 were isolated from C. zuccariniana, 5 and 6 from C. achaia, 7 from C. thessala ssp. drakiensis and compounds 8 and 9 from C. deusta. Compound 1, 8α-O-(3,4-dihydroxy-2-methylenebutanoyloxy)-dehydromelitensine was found to be the most active, exhibited a considerable growth inhibiting activity against three of the cell lines tested, while compound 5 8α-O-(3-hydroxy-2-methylenepropanoyl)dehydromelitensin exhibited a growth inhibiting effect against most of the tested cell lines. A new eudesmanolide (8α-hydroxysonchucarpolide, 4) was isolated from C. zuccariniana and its structure was elucidated by spectroscopic methods.

The chemical composition of the essential oils of twenty-five populations of Cistus creticus subsp. creticus L. from the island of Crete (Greece) and their interpopulation variability were analysed in detail by GC-MS. 142 compounds were identified representing an average of 56.8-89.8% of the oil composition. The components are represented here by homologous series of monoterpenes, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes, diterpenes, labdane diterpenes, aldehydes, alkanes, esters, fatty acids, ketones, and others. Labdane diterpenes were detected and identified in the essential oils and have been found in high percentage composition. The results from the chemical analysis of the essential oils were submitted to chemometric cluster analysis in order to detect some pattern distribution and to identify which constituents can differentiate the groups of individuals. Two main chemotypes (clusters) were well differentiated; the first deals with eight populations of West Crete and the second with the rest of the populations. Cluster analysis based on labdane type diterpenes patterns, proved to be the best chemotype for the examined populations among the other chemical groups.

There is confusion in the literature related to the characterization of biologically important C13 manoyl oxide epimers by using their mass spectrometric data. Furthermore a method for calculating the epimeric purity has not been established. In this work mixtures of manoyl oxide C13 epimers and pure ent-13-epi-manoyl oxide have been isolated from plant extracts. The GC-MS analysis allows the characterization of each stereoisomer in a sample of natural origin: the ratio of intensities of peaks m/z 275: 257 is lower in ent-13-epi-manoyl oxide than in manoyl oxide. NMR spectroscopy is used to give experimental evidence and simple calculations were performed to support the MS data. On the basis of these results the characterization of the two stereoisomers and the calculation of their ratio in plant extracts and essential oils can be done in a routine basis. The biological activity evaluation of mixtures with different epimeric composition of manoyl oxide showed that the ratio of the two epimers is important for their antibacterial activity. Ent-13-epi-manoyl oxide seems to be more active than its epimeric congener against Gram-positive bacteria. © 2002 Elsevier Science Ltd. All rights reserved.

The oils of the aerial parts of Acinos suaveolens, collected in two different regions in Greece were analyzed by GC/MS. Both oils were found to be dominated by oxygenated monoterpenes. The main compound in both samples was pulegone 67.7% and 77.7%, respectively. Furthermore, the bacteriostatic activity of the oils against three Gram positive and two Gram negative bacteria was evaluated. © 2002, Taylor & Francis Group, LLC. All rights reserved.

The composition of the essential oil from the leaves of Cistus monspeliensis (Cistacea) shows seasonal and diurnal variation. The essential oils were investigated by GC-FID and GC-MS. Labdanes which were identified for the first time in the essential oil of C. monspeliensis, are predominant in autumn and in winter and they were found in great amount with clerodanes in spring. Manoyl oxide, 3β-hydroxy-13-epi-manoyl oxide, 3β-acetoxy-13-epimanoyl oxide and their isomers varied through the day and the year. The results obtained fro GC-MS analysis of the oils were submitted to statistical analysis. © 2002 Elsevier Science Ltd. All rights reserved.

Differential scanning calorimetry has been employed to study the thermal effects of vinblastine sulfate upon aqueous, single and multiple bilayer dispersions of 1,2-dipaimitoyl-3-sn-phosphatidylcholine (DPPC). The calorimetric results summarized to an increase in the gel to liquid-crystalline phase transition enthalpy and the abolishment of the L’(beta) (gel phase) to P’(beta) (ripple phase) pretransition for the uni- and multilamellar dispersions, as well as an increase in the transition temperature T-m and the transition cooperativity for single bilayer DPPC/vinblastine mixed vesicles, are consistent with an induced, partially interdigitated, gel phase. Computational analysis has been successfully applied to clarify the intermolecular effects and verify the feasibility of the proposed interdigitation for the vinblastine sulfate molecules and also for the ursodeoxycholic acid (UDCAH) and bromocylated taxanes, which have been shown to induce an interdigitated gel phase in DPPC bilayers. (C) 2002 Elsevier Science B.V. All rights reserved.

Liposomes prepared from lipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) with cholesterol were used to investigate the percentage of vinblastine encapsulation and the influence of lipid composition on the retention properties of vinblastine in buffer as well as in cell culture medium. Differential scanning calorimetry (DSC) was applied, to study the effect of cholesterol on the phase transition temperature and on the ΔH of the two liposome formulations. The cytotoxic and cytostatic activity of the liposome-encapsulated vinblastine was also examined against six leukaemic human cell lines. The results showed that encapsulation of vinblastine into liposomes was greater than 98% with a drug-phospholipid molar ratio of 0.13-0.18. The major improvement in vinblastine retention in buffer as well as in culture medium was achieved by employing DPPG. The DSC data showed that vinblastine exerted a more perturbing effect in DPPC-cholesterol bilayers than in DPPG-cholesterol bilayers and this may explain their lower retention time. The 50% growth-inhibiting (GI50) and cytostatic (TGI) activity of liposomal vinblastine did not seem to be affected by the type of the liposome while the 50% cytotoxic activity (LC50) was affected in four out of the six cell lines tested. The parameters GI50, TGI and LC50 were estimated according to the instructions given by the NCI.

The walnut oil (Juglans regia L.) total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and they were analyzed by high performance thin layer chromatography (HPTLC) /FID and GC-MS. The oil was found to be rich in neutral lipids (96.9% of total lipids) and low in polar lipids (3.1% of total lipids). The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of sphingolipids. GC-MS data showed that the main fatty acid was linoleic acid. Unsaturated fatty acids were found as high as 85%, while the percentage of the saturated fatty acids was found 15%. Two types of liposomes were prepared from the isolated walnut oil phospholipids and characterized as new formulations. These formulations may have future applications for encapsulation and delivery of drugs and cosmetic active ingredients.

The chemical composition of the essential oils obtained from the leaves and bracts of hydroponically cultivated Origanum dictamnus were analyzed by GC-MS techniques. Three different concentrations of phosphorus (5, 30, and 60 mg/L) in the nutrient solution were used for the cultivation, using the nutrient film technique (NFT). A total of 46 different compounds were identified and significant differences (qualitative and quantitative) were observed between the samples. Carvacrol and p-cymene were identified as the main compounds in all samples analyzed, whereas thymoquinone was found in higher percentage in the leaves than in bracts. The essential oils were tested for their antibacterial activity against Gram-positive and Gram-negative bacteria. The oils obtained from the bracts were found to be more active. The results obtained from GC-MS analyses were submitted to chemometric analysis.

The composition of the essential oils of fifteen populations of Cistus salviifolius L. (Cistaceae) from Crete (Greece) and their interpopulation variability was investigated by GCMS. 167 compounds were identified representing an average of 96-100% of the oil composition. Labdane diterpenes were detected and identified in the essential oils of Cistus salviifolius L., for the first time. The results obtained from GC-MS analysis of the oils were submitted to a Principal Component Analysis (PCA). Three main chemotypes (clusters) were differentiated. All the essential oils were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria. Only chemotype III having camphor as a major constituent was active against Gram-positive bacteria. Camphor, viridiflorol, longiborneol, phyllocladene, abietatriene and cis-feruginol are the main constituents, while the group of oxygenated sesquiterpenes has the highest percentage composition. © 2002 Elsevier Science Ltd. All rights reserved.

Sclareol (1) and ent-3 beta -hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds I and 3 were found to induce apoptotic cell death in human T cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds I and 3 in these T cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used. (C) 2001 Elsevier Science Ltd. All rights reserved.

The terpenoids are a class of natural products with biological activity. The number of isoprene units from which they are biogenetically composed is used for their classification. The diterpenes are another group from which several compounds with high biological activity have been produced. The most studied plant families are Asteraceae and Labiatae, as well as Conifers, which are the main sources of diterpenes. The lack of, or insufficient, chromatographic data (RI, Rt) as well as of reference compounds for most labdanes is the main reason for their absence from the chemical fingerprints of plantextracts. Also their use as chemotaxonomic markers within the species in which they occur is limited. The configuration at the C-13 carbon atom has been investigated, especially in the case of manoyl oxide, as well as the various isomers, the ratio of which determines antimicrobial activity. A variety of biological activities have been encountered in labdane diterpenes such as antibacterial, antifungal, antiprotozoal, enzyme inducing, anti-inflammatory activities and modulation of immune cell functions. More recent studies have shown that labdane exhibits significant cytotoxic and cytostatic effects against leukemic cell lines of human origin and interferes with the biochemical pathways of apoptosis and the cell cycle phases, as well as with the expression of several protooncogenes such as c-myc and bcl-2. This report underlines the role of these compounds, not only as toolsfor the study of the biochemistry and regulation of biochemical and metabolic pathways of mammalian cellular systems, but also as potential pharmacological agents in the fight against diseases such as cancer and heart disorders. © 2001 Elsevier B.V. All rights reserved.

The composition of the essential oil obtained from different parts of Crithmum muritimum L., growing in Amorgos island (Greece) and their interpopulation variability were investigated by GC and GUMS. The yield of the essential oil from various parts of the plants in the examined populations, varied from 0.08-0.99%. Monoterpenes constituted the more important portion of the oil while a few sesquiterpene hydrocarbons were detected in small amounts. The major compounds detected in all cases were p-cyrnene (0.8-15.8%), methyl thymol (4.4-41.0%), terpinen-4-01 (3.9-16.0%), sabinene (1.1-24.7%), γ-phellandrene (0-10.3%), β-terpinene (6.9-28.6%), or even dillapiole (0-48.8%) in one case. The results obtained from GUMS analysis of the oils were subjected to multivariate analysis (cluster analysis). © 2001, Taylor & Francis Group, LLC. All rights reserved.

Essential oils and hexane extracts from Cistus monspeliensis L. leaves and fruits were analysed by GC-MS. Manoyl oxide and its isomers, 3β-hydroxy-manoyl oxide, 3β-hydroxy-13-epi-manoyl oxide, as well as 3β-acetoxy-13-epi-manoyl oxide were detected for the first time in C. monspeliensis L. Ent-13-epimanoyl oxide was isolated from the hexane extract of leaves and its structure was determined using spectroscopic methods. In vitro cytotoxic activity of ent-13-epi-manoyl oxide and mixtures of manoyl oxide isomers at 10-4 M concentrations ranged from 11.1 to 32.2% of the activity for a vinblastine control evaluated against nine leukemic cell lines. The results showed that the manoyl oxide isomer mixtures as well as ent-13-epi-manoyl oxide exhibited a slight growth inhibiting activity.

The 1-O-hexadecyl-2-O-methyl-sn-glyceryl phosphodiester AZT 4 and hexadecyl-phosphodiester AZT 5 derivatives were synthesized and found to be active against HIV-1, HIV-2, and tumor cell proliferation. Compared to AZT, compound 4 possessed ca. 10-fold lower anti-HIV activity and ca. 10-fold higher anti-tumor cell activity. Compound 5 was 10-fold less potent than compound 4 in both biological tests. In an attempt to correlate biological activity of compounds 4 and 5 with structure, their conformational and thermal effects on membrane bilayers were compared using a combination of NMR spectroscopy, computational analysis, and Differential Scanning Calorimetry. The obtained results showed that compound 4 adopts a compact conformation in which the alkyl chain, the 2-methoxyglyceryl functionality, and the methyl group of thymine are in spatial proximity, while analogue 5 possesses a less compact conformation of the nucleoside base and the alkyl chain. The presence of the 2-methoxyglyceryl group in compound 4 may augment its potency by inducing a turn of the alkyl chain stabilized by hydrophobic interactions. The DSC scans show that conjugate 4 affects less effectively the thermotropic properties of model membrane bilayers than compound 5. This may be attributed to the fact that compound 4 is incorporated in a compact conformation and does not perturb significantly the trans:gauche isomerization of the membrane phospholipids. In contrast, conjugate 5 may enter with a less compact conformation and perturb more the membrane bilayers.

The composition of the essential oils of nine populations of Cistus parviflorus L., from Crete (Greece) and their interpopulation variability, were investigated by GC-MS. 114 compounds were identified representing an average of 85-96% of oil composition. Labdane diterpenes were detected and identified in the essential oils of Cistus parviflorus L. for the first time. The results obtained from GC-MS analysis of the volatile oils were submitted to principal component and chemometric cluster analysis. Two main chemotypes (clusters) were differentiated; the first of West Crete and the second of East Crete. Carvacrol, caryophyllene oxide, α-epi-cadinol, abietatriene, 4-epi-dehydroabietol, dehydro abietol, cis-ferruginol and manoyl oxide mixture of isomers are the main constituents, while oxygenated sesquiterpenes as well as diterpenes have been found in high percentage composition. © 2001 Elsevier Science Ltd.

The chemical composition of the oils from two endemic and rare taxa of the genus Origanum L., i.e O. calcaratum Juss. = O. tournefortii Aiton and O. scabrum Boiss. et Heldr., from Greece, were investigated by GC and GC/MS. Twenty-two compounds were identified into the oil of O. calcaratum, representing 98.0% of the oil composition. The main compounds were p-cymene (18.1%), γ-terpinene (9.6%), thymol (42.8%), carvacrol (12.9%) and isocaryophyllene (4.7%). In the oil of O. scabrum, 28 compounds were identified, representing 98.0% of the oil composition. The main compound was carvacrol (66.7%), while p-cymene (7.8%), γ-terpinene (3.6%), caryophyllene oxide (2.1%) and manool (2.7%) were found in large amounts. The oils were investigated for their antimicrobial activity against Gram-positive, i.e., Staphylococcus aureus, S. epidermidis and Gram-negative, i.e., Escherichia coli, Klebsiella pneumoniae microorganisms. © 2001, Taylor & Francis Group, LLC. All rights reserved.

The membrane perturbing 3, 7, 4′, 5′-tetramethyl ether of myricetin 1 was isolated from Cistus monspeliensis L. Its structure was elucidated and its conformational properties were explored using a combination of 2D NMR spectroscopy and computational chemistry. The obtained results showed that compound 1 adopts four enantiomeric pairs of low energy conformers characterized: (a) by an aromatic ring B twisted through rotation about C2-C1′ bond from the rigid isoflavone ring; (b) a 4′-O-CH3 bond oriented out of the plane with equal probability upwards or downwards the phenyl ring B, while all the other O-CH3 bonds are oriented in the plane of the aryl ring. Two of these enantiomeric pairs are lowest in energy. These possible bioactive conformers are possibly stabilized by van Der Waals interactions. The 3′, 5-diacetyl derivative 2 of compound 1 was synthesized and its structure elucidation was achieved based on the chemical shift assignment of the parent compound 1. The Differential Scanning Calorimetry (DSC) results revealed that the degree of the thermal effects exerted by the flavonoids at dipalmitoylphosphatidyl choline (DPPC) bilayers followed the order 1 > 2 > myricetin. Their antimicrobial activity against Gram positive bacteria followed the same order.

Two labdane type diterpenes, labd-13(E)-ene,-8 alpha ,15-diol (1) and labd-13(E)-ene,-8 alpha ,15-yl acetate (2) were isolated from the hexane extract of Cistus creticus subsp. eriocephalus (Viv.) Greuter & Burdet leaves, while (+)-19-acetoxy-cis-clerodan-3-en-15-oic acid (3) was isolated from the hexane extract of Cistus monspeliensis L. leaves. The compounds were examined for their in vitro cytostatic and cytotoxic activity against nine human leukemic cell lines, three of which exhibited a multidrug resistant phenotype. They were also evaluated for their anti-inflammatory activity in vivo on the back of hairless mice. The cytostatic and cytotoxic activity of the tested diterpenes followed the order 1 >2 >3. Topical application of the diterpenes on barrier disrupted skin did not seem to have a significant contribution to the repair rate of the skin barrier.

The structure of a novel CC clerodane type diterpenoid, namely (+)-19-acetoxy-cis-clerodan-3-ene-15-oic acid 1 was elucidated and its conformational properties were explored using a combination of high field NMR spectroscopy and computational analysis. The structural analysis provided results consistent with those obtained by a single X-ray diffraction study of its dicyclohexylammonium salt. The new clerodane type diterpene isolated in large quantities from Cistus monspeliensis L. leaves was found to exhibit significant antibacterial activity against Staphyloccoci (MIC50 = 0.085 mM) and therefore represents a promising lead compound. Interestingly the deacetylated derivative 2 of compound 1 was inactive. © 2001 Elsevier Science Ltd.

Two myricetin derivatives, the 3,7,4',5'-tetramethyl ether of myricetin (1), isolated from the hexane extract of Cistus monspeliensis, and its 3',5-diacetyl derivative (2) which was synthesized, and the parent compound, myricetin (3), were examined for their in vitro cytotoxic activity against nine human leukemic cell lines, two of which were mdr cell lines. Compound 2 exhibited higher cytostatic and cytotoxic activities in comparison to compound 1, while compound 3 was inactive against all tested cell lines. Vinblastine was used as a control. (C) 2000 Academic Press.

The lipids of the stratum corneum are considered responsible for the most important functions of the skin, such as the transepidermal water loss, as well as the transdermal penetration of the chemical substances. Topical application of lipids similar to the physiological stratum corneum (SC) on barrier disrupted skin, could enhance the recovery rate of the skin barrier. A mixture of natural lipids or liposomes with the same lipid composition, were applied and their pharmacological action was investigated. The tests were done in vivo, on the back of hairless mice. Comparative results were obtained and showed that the liposomes had a higher turnover of the skin barrier in contrast to that of the mechanical mixture of lipids. (C) 2000 Academic Press.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [3H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control.

The chemical composition of the essential oils of the flowering parts and of the hexane extracts of the leaves, of three species of the genus Ferulago (Apiaceae) from Greece, i.e., F. thyrsiflora, F. sylvatica and F. nodosa, was analysed by GC and GC-MS. Manoyl oxide isomers and lupanine, were identified in the hexane extracts of F: thyrsiflora. Dillapiole, was identified in all hexane extracts. Ent-3 beta-hydroxy-13-epi-manoyl oxide was identified and isolated from the essential oil of F. thyrsiflora. All these compounds are new reports for the genus Ferulago and are useful chemotaxonomic markers. Chemical data of F. thyrsiflora is presented for the first time. The essential oils and the extracts were investigated for their antimicrobial activity against Gram-positive and Gram-negative microorganisms.

Ent-3 beta-hydroxy-13-epi-manoyl oxide (1), was converted to its thiomidazolide derivative (2) which was tested for its cytotoxic activity against a panel of established human leukemic cell lines. Compound 2, exhibited cytotoxic activity against 13 of the cell lines tested. Additionally, compound 2 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis and on cell proliferation. The morphology of the cells and the kind of death induced, was investigated. Flow cytometry experiments on a leukemic cell line was also performed. The results show that the semi-synthetic compound, showed a significant antiproliferative effect and kills cells through the process of apoptosis. The appearance of the apoptotic signs was time and dose dependent. From the flow cytometry experiments, a synchronisation through a delay of the cells in G0/1, phase seems to take place. Anticancer Res

The qualitative and quantitative analysis of labdane-type diterpenes of the hexane extracts and of the essential oils of the leaves, fruits and resin 'Ladano', of Cistus creticus subsp. creticus and Cistus creticus subsp. eriocephalus, have been carried out by GC and GC-MS analysis using two capillary chromatographic columns, i.e., HP-5MS and CP-Wax. The methanolic extract of the fruits of C. creticus subsp. creticus was examined and seven labdane diterpenes were isolated and identified by spectroscopic methods. Data on the investigation of labdane diterpenes by GC and GC-MS is limited and most of them have never been analysed by this method. The results obtained by this analysis could be useful for identifying them in crude plant extracts. Manoyl oxides were studied further for the percentage content of their isomers. The hexane extracts of the two subspecies as well as the manoyl oxide isomers isolated from the methanolic extract of the fruits of C. creticus subsp. creticus, were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria. Global numerical differences of these C. creticus subspecies, based on labdane diterpenes content in the hexane extracts as well as in the essential oils, were established by statistical methods. Phenotypic differences are discussed.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 mu g/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 mu g/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G(0/1) seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis. (C) 1999 Elsevier Science Ltd. All rights reserved.

The peracetylated derivative of kaempferol-3-O-beta-D-(6 “-E-p-coumaroyl) glycopyranoside (tiliroside) (1a) was tested for its cytotoxic and cytostatic activity against several human leukemic cell lines. The significant cytotoxic activity of this derivative, prompted to an additional examination on some of the cell lines used. The effect on the uptake of [H-3]thymidine as a marker of DNA synthesis and on the cell proliferation, was investigated as well as the morphology of the cells and the kind of death induced, using the Wright-Giemsa dye and horizontal agarose-gel electrophoresis. Flow cytometric experiments of 1a on some leukemic cell lines was also performed. Compound 1a showed a significant antiproliferative effect as soon as 1 h of continuous incubation at all cell lines tested. Cells were killed, through the process of apoptosis and the appearance of the apoptotic signs was time and dose-dependent, while from the flow cytometric experiments, a synchronisation (through a delay probably in the G(0/1) phase) of the cells seems to take place. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.

The chemical composition of the volatile constituents from bracts and leaves of wild and hydroponically cultivated Origanum dictamnus were analysed by GC and GC-MS. Three different levels of nitrogen (100, 150, 200 mg/l), were used in the nutrient solution for the cultivation, using the Nutrient Film Technique (N.F.T). Carvacrol was the predominant compound in all cases. The essential oils were investigated for their antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Escherichia coli, and Pseudomonas aeruginosa.

Fractionation of the resin Ladano from Cistus creticus subsp. creticus and susceptibility testing using the chromatographic fractions showed that its antistaphylococcal activity was mainly due to the diterpene sclareol. The antimicrobial activity of its essential oil, of the chromatographic fractions, and of the isolated compounds was also evaluated against Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus hominis.

The essential oils from flower heads and disc florets of wild Chamomilla recutita (L.) Rausch., collected in Attica (Greece), were obtained by hydrodistillation. With the use of GC and GC/MS, 21 components were identified in both oils. The major constituents, were (E)-β-farnesene (21.2% and 5.6%) and bisabolone oxide A (16.0% and 9.0%) in the oils of the flower heads and disc florets, respectively. © 1998, Taylor & Francis Group, LLC. All rights reserved.

Nine labdane type diterpenes isolated from the plant Cistus creticus subsp. creticus and from the resin 'Ladano' which is excreted on the surface of the leaves and stems of this plant, were examined for their in vitro cytotoxic activity against 14 human leukemic cell lines. Compound 1, (13E)- labd-13-ene-8α, 15-diol, exhibited cytotoxic activity against 13 of the cell lines tested, while compound 2, (13E)-labd-7,13-dienol, was active only against HL60 cells. Further compound 1 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis.

The total methanol extract and chromatographic fractions of the extract of the aerial parts of Hypericum perforatum L. were investigated for their antiviral activity against human cytomegalovirus (HCMV) AD169 laboratory strain by modification of the widely used diagnostic method ‘shell-vial’ culture. The plant selection was based on its traditional use against viruses of the Herpes family in Greece. The results showed extract fraction III had the most potent antiviral activity against HCMV which was also superior to that of ganciclovir. (C) 1998 John Wiley & Sons, Ltd.

The chemical composition of the essential oil of the leaves of Cistus creticus subsp. eriocephalus (Viv.) Greuter and Burdet, (Cistaceae), was investigated by GC/MS. Thirty-nine components were identified, representing 73.9% (based on % total peak area by GC) of the oil composition. The main components of the oil were α-cadinene (6.5 %), δ-cadinene (6.5 %), viridiflorol (5.4%), bulnesol (6.3%), ledol (3.8%), α-copaene (3.8%), β-selinene (3.4%), cubenene (3.3%), manoyl oxide (9.9%) and 13-epi-manoyl oxide (3.4%). Antibacterial studies were carried out in vitro against Gram-positive and Gram-negative organisms.