Chemical Constituents and Anticancer Activities of the Extracts from Phlomis × commixta Rech. f. (P. cretica × P. lanata).

The present work is the first report on the ingredients of the P. × commixta hybrid, a plant of the genus Phlomis. So far, thirty substances have been isolated by various chromatographic techniques and identified by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS. The compounds are classified as flavonoids: naringenin, eriodyctiol, eriodyctiol-7-O-ß-D-glucoside, luteolin, luteolin-7-O-ß-D-glucoside, apigenin, apigenin-7-O-ß-D-glucoside, diosmetin-7-O-ß-D-glucoside, quercetin, hesperetin and quercetin-3-O-ß-D-glucoside; phenylpropanoids: martynoside, verbascoside, forsythoside B, echinacoside and allysonoside; chromene: 5,7-dihydroxychromone; phenolic acids: caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, chlorogenic acid methyl ester, gallic acid, p-coumaric acid and vanillic acid; aliphatic hydrocarbon: docos-1-ene; steroids: brassicasterol and stigmasterol; a glucoside of allylic alcohol, 3-O-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranosyl-oct-1-ene-3-ol, was fully characterized as a natural product for the first time. Two tyrosol esters were also isolated: tyrosol lignocerate and tyrosol methyl ether palmitate, the latter one being isolated as a natural product for the first time. Moreover, the biological activities of the extracts from the different polarities of the roots, leaves and flowers were estimated for their cytotoxic potency. All root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines.

Isolation of Secondary Metabolites from Achillea grandifolia Friv. (Asteraceae) and Main Compounds' Effects on a Glioblastoma Cellular Model.

This study aims at the isolation and structural determination of the secondary metabolites of the herbaceous perennial plant Achillea grandifolia Friv. (Asteraceae). The examination of the non-volatile content of the leaves and flowers of A. grandifolia afforded the isolation of sixteen secondary metabolites. On the basis of NMR spectra, the identified compounds included ten sesquiterpene lactones; three guaianolides-rupicolin A (1), rupicolin B (2), and (4S,6aS,9R,9aS,9bS)-4,6a,9-trihydroxy-9-methyl-3,6-dimethylene-3a,4,5,6,6a,9,9a,9b-octahydro-3H-azuleno [4,5-b]furan-2-one (3); two eudesmanolides-artecalin (4) and ridentin B (5); two sesquiterpene methyl esters-(1S,2S,4αR,5R,8R,8αS)-decahydro-1,5,8-trihydroxy-4α,8-dimethyl-methylene-2-naphthaleneacetic acid methylester (6) and 1ß, 3ß, 6α-trihydroxycostic acid methyl ester (7); three secoguaianolides-acrifolide (8), arteludovicinolide A (9), and lingustolide A (10); and an iridoid-loliolide (11). Moreover, five known flavonoids, i.e., apigenin, luteolin, eupatolitin, apigenin 7-O-glucoside, and luteolin 7-O-glucoside (12-16) were also purified from the aerial parts of the plant material. We also investigated the effect of rupicolin A (1) and B (2) (main compounds) on U87MG and T98G glioblastoma cell lines. An MTT assay was performed to define cytotoxic effects and to calculate the IC50, while flow cytometry was employed to analyze the cell cycle. The IC50 values of reduced viability during the 48 h treatment for compound (1) and (2) were 38 µM and 64 µM for the U87MG cells and 15 µM and 26 µM for the T98G cells, respectively. Both rupicolin A and B induced a G2/M cell cycle arrest.

Exploring the Medicinal Potential of Achillea grandifolia in Greek Wild-Growing Populations: Characterization of Volatile Compounds, Anti-Inflammatory and Antioxidant Activities of Leaves and Inflorescences.

Various species of the genus Achillea L. (Asteraceae) are traditionally used worldwide for wound healing against diarrhea, flatulence, and abdominal pains, as diuretic and emmenagogue agents. In the present study, the essential oils (EOs) obtained separately from the leaves and inflorescences of wild-growing Achillea grandifolia Friv. from Mt. Menoikio and Mt. Pelion (Greece) were analyzed by Gas Chromatography-Mass Spectrometry. The major compounds found in EOs of A. grandifolia inflorescences from Mt. Menoikio were as follows: cis-thujone (36.9%), 1,8-cineole (11.9%), camphor (10.0%), ascaridole (7.3%), α-terpinene (6.4%), sabinene (4.1%), trans-thujone (3.6%), and cis-jasmone (3.4%). In leaves from Mt. Menoikio, they were as follows: cis-thujone (50.8%), 1,8-cineole (20.0%), trans-thujone (5.5%), camphor (5.5%), borneol (3.6%), and α-terpineol (3.1%). In inflorescences from Mt. Pelion, they were as follows: camphor (70.5%), camphene (5.9%), cis-jasmone (3.2%), bornyl acetate (3.2%). In leaves from Mt. Pelion, they were as follows: camphor (83.2%), camphene (3.9%), and borneol (3.7%). Subsequently, the samples were first time tested for their antioxidant activities with the interaction of EOs with DPPH (2,2-diphenyl-1-picrylhydrazyl) and their inhibition of lipid peroxidation, as well as for their anti-inflammatory activity through the soybean LOX (lipoxygenase) inhibition. All of the examined samples were found effective. A. grandifolia leaves presented the highest antioxidant potential according to the DPPH method, and the highest percentage of LOX inhibition. The study herein investigated for the first time the leaves and the inflorescences of A. grandifolia separately, and the results generally align with similar studies from neighboring countries (Turkey and Serbia) in terms of the yields and categorization of main EO compounds (oxygenated monoterpenes). However, the findings were not in agreement with previously studied Greek material, as a higher amount of cis-thujone and lower antioxidant activity are reported herein. Both the EOs of inflorescences and the leaves of the wild-growing population collected from Mt. Menoikio were characterized by a high quantity of cis-thujone (36.9% and 50.8%, respectively).

Therapeutic Potential of Linearol in Combination with Radiotherapy for the Treatment of Glioblastoma In Vitro.

Glioblastoma is one of the most malignant and lethal forms of primary brain tumors in adults. Linearol, a kaurane diterpene isolated from different medicinal plants, including those of the genus Sideritis, has been found to possess significant anti-oxidant, anti-inflammatory and anti-microbial properties. In this study, we aimed to determine whether linearol could exhibit anti-glioma effects when given alone or in combination with radiotherapy in two human glioma cell lines, U87 and T98. Cell viability was examined with the Trypan Blue Exclusion assay, cell cycle distribution was tested with flow cytometry, and the synergistic effects of the combination treatment were analyzed with CompuSyn software. Linearol significantly suppressed cell proliferation and blocked cell cycle at the S phase. Furthermore, pretreatment of T98 cells with increasing linearol concentrations before exposure to 2 Gy irradiation decreased cell viability to a higher extent than linearol or radiation treatment alone, whereas in the U87 cells, an antagonistic relationship was observed between radiation and linearol. Moreover, linearol inhibited cell migration in both tested cell lines. Our results demonstrate for the first time that linearol is a promising anti-glioma agent and further studies are needed to fully understand the underlying mechanism of this effect.

Exploring the Ecological Preferences and Essential Oil Variability in Wild-Growing Populations of the Endangered Local Greek Endemic Thymus holosericeus (Lamiaceae).

Thymus holosericeus Celak. (Lamiaceae) is a taxonomically isolated and endangered local endemic thyme species which is geographically isolated in four Ionian Islands (West Greece). The present study investigated the essential oil (EO) composition, the ecological preferences, and their correlations in three T. holosericeus wild-growing populations from Zakynthos (ΤH-Z), Cephalonia (ΤH-C) and Lefkada (ΤH-L). The variations in essential oil yield and the composition of T. holosericeus populations were evaluated using hydrodistillation, GC/MS, TLC and NMR analysis. The climatic conditions of each sample were organized and analyzed in RStudio with the raster package and in SPSS with Pearson's Canonical Correlation Analysis (CCA), respectively. The aerial parts of the plants varied in EO yields from 1.92 to 2.28% w/v. The analysis of EO constituents revealed noteworthy qualitative and quantitative inter-population variation. The composition of EOs revealed the presence of linalool (82.77%) and borneol (5.95%) as major compounds in ΤH-Z, while carvacrol (35.34%), geraniol (23.98%), linalool (14.37%), borneol (5.66%), thymol (4.27%) and p-cymene (4.08%) were the main compounds in ΤH-C and linalool (40.37%), geraniol (39.42%) and borneol (5.20%) were dominant components in ΤH-L. The results of the gas chromatography procedure have also been confirmed by 1H and 13C-NMR and TLC analysis. The ecological profile showed an average annual precipitation of 942 ± 18.33 mm and the temperature limits in which T. holosericeus seems to adapt to are 6.80± 1.08 °C 27.70 ± 0.70 °C. Regarding the examined samples, TH-C was adapted to the driest summer and coldest winter conditions, TH-Z was adapted to the lowest annual precipitation with the most complex climatic conditions, and TH-L was adapted to the highest summer temperatures with the lowest precipitation in the wettest period of the year. For each sampled population, the CCA identified the association of the samples' EOs composition with the prevailing local environmental conditions.

Ganoderma adspersum (Ganodermataceae): Investigation of Its Secondary Metabolites and the Antioxidant, Antimicrobial, and Cytotoxic Potential of Its Extracts.

Ganoderma is a genus of wood-degrading mushrooms with medicinal importance. Most Ganoderma species have been studied extensively for their secondary metabolites, biological activities, and ecological value. In this study, the biological activities of the extracts of G. adspersum growing wild on Morus alba trees in the region of Western Thrace (Greece) were evaluated, and the petroleum ether, dichloromethanolic, and methanolic extracts were studied further for their secondary metabolites. Six substances were isolated by chromatographic (Clumn Chromatography (C.C.), High Performance Liquid Chromatography (HPLC)) and spectroscopic methods (Nuclear Magnetic Resonance (NMR)), which were classified in the following categories: (a) unsaturated fatty acids: cis-oleic acid (1); (b) sterols: ergosta-7,22-dien-3-one (2), ergosta-7,22-dien-3-ol (3), and ergosta-5,7,22-trien-3-ol (4); and (c) lanostane-type triterpenoids: applanoxidic acid G (5) and applanoxidic acid A (6). Finally, the biological activities of the extracts were estimated for their antioxidant, antimicrobial, and cytotoxic potential. The methanolic extract of G. adspersum showed the highest total antioxidant activity. The results of the antimicrobial activities indicated that all of the extracts had a minimum inhibitory concentration (MIC) ranging between 39.1 and 312.5 µg/mL. The evaluation of the cytotoxic activity of the samples showed once again that the methanolic extract was the most potent among the examined extracts, with half-maximal inhibitory concentration (IC50) values of 19.22 µg/mL (Hep2c cells), 32.9 µg/mL (RD cells), and 8.94 µg/mL (L2OB cells). Moreover, the bioactivity scores of the isolated secondary metabolites were calculated using the online computer software program Molinspiration. The compounds showed promising bioactivity scores for drug targets.

Chemical constituents isolated from the aerial parts of Helleborus cyclophyllus (A. Braun) Boiss. (Ranunculaceae), evaluation of their antioxidant and anti-inflammatory activity in vitro and virtual screening of molecular properties and bioactivity score.

Chemical investigation of ethyl acetate extract from the aerial parts of Helleborus cyclophyllus (A.Braun) Boiss. led to the isolation of ten natural products, and their structures were identified to be: 2-deoxy-D-ribono-1,4-lactone (1), 2-O-feruloyl-L-malate (2), three flavonoids: quercetin 3-O-ß-D-galactopyranoside (3), quercetin 3-O-6''-(3-hydroxy-3-methyl-gloutaryl)-ß-D-glucopyranoside (4) and quercetin 3-O-(2‴-caffeoylsophoroside) (5), 6-O-caffeoyl-1-O-p-coumaroyl-ß-D-glucopyranoside (6), two ecdysteroids: 20-hydroxyecdysone (7) and polypodine B (8) and two bufadienolides: deglucohellebrin (9) and hellebrin (10), on the basis of MS and NMR spectra. This is the first report on the occurrence of compounds (2)-(6) in the genus Helleborus. The chemotaxonomic significance of these compounds was summarised. Bioactivity score, molecular and pharmacokinetic properties of the isolated compounds were calculated by online computer software program Molinspiration. Compounds showed promising bioactivity scores for drug targets. Moreover, some of the isolated phenolic compounds were tested for their antioxidant and antiinflammatory activities. Tested compounds presented antioxidant ability correlated to the presence of the phenolic hydroxyl groups.

Effects of Cornus and Its Mixture with Oregano and Thyme Essential Oils on Dairy Sheep Performance and Milk, Yoghurt and Cheese Quality under Heat Stress.

The effect of a diet supplemented with a novel cornus extract, enriched with essential oils of oregano and thyme, on the performance of Chios cross-bred dairy sheep was investigated during the summer period. The plant extracts were prepared using a "green" method based on aqueous extraction. A total of 45 lactating ewes were allocated into three equal groups in a randomized block design. The three groups were fed the same feed allowance, roughage based on Lucerne hay and wheat straw and a concentrate based on cereals and oil cakes (the control diet). The diet of two groups was fortified with cornus extract, with or without oregano and thyme essential oils, at a level 0.515 g of plant extract/essential oils per kg of concentrate. Individual milk yield was recorded weekly and feed refusals were recorded on a pen basis daily, during a six-week period of lactation. Milk samples were analyzed for the chemical composition of protein, fat, lactose and solids-not-fat constituents, somatic cell counts and total viable bacteria counts. Moreover, the milk of each group was used for yoghurt and Feta cheese production. The lipid oxidative stability, protein carbonyl content and fatty acid composition of milk, yoghurt and cheese samples were also evaluated. The results showed that the incorporation of novel plant extracts and essential oils increased the milk production per ewe. Dietary supplementation with cornus extracts and essential oils lowered lipid and protein oxidation in milk, yoghurt and cheese samples, compared to the control. However, diet supplementation with herbal extracts did not affect the fatty acid profile in milk, cheese and yoghurt or the serum biochemical parameters. In conclusion, dietary supplementation with cornus in combination with oregano and thyme has the potential to improve feed utilization and the performance of high-yield dairy Chios cross-bred ewes reared under heat stress.

Deglucohellebrin: A Potent Agent for Glioblastoma Treatment.

BACKGROUND: Glioblastoma is the most common primary brain tumor in adults with a dismal prognosis. To date, several anticancer agents have been isolated from plants. Helleborus odorus subsp. Cyclophyllus is an endemic plant of the Balcan flora. Herewith, we investigated for the first time, the anti-glioma effect of deglucohellebrin (DGH) extracted from the roots of Helleborus. METHODS: We investigated the effect of DGH in U251MG, T98G and U87G glioblastoma cell lines. We selected the T98G cells because of their inherent temozolomide resistance. RESULTS: The IC50 value of reduced viability for DGH was 7x10-5M in U251MG cells, 5x10-5M for the T98G cells and 4x10-5M in U87G cells during 72h treatment. DGH induced G2/M cell cycle arrest, caspace-8 activation and significant mitochondrial membrane depolarization, suggesting the activation of the intrinsic, mitochondrial- dependent apoptotic pathway. DGH and temozolomide induced changes in CDs' expression in U251MG and T98G cells. In zebrafish, DGH did not induce toxicity or behavioral alterations. CONCLUSION: The present study is the first to determine the anti-glioma activity of DGH. DGH may be a potent agent for glioblastoma treatment and further studies are needed.

Antioxidant Activity and Chemical Composition of Essential Oils of some Aromatic and Medicinal Plants from Albania.

The chemical compositions have been investigated of the volatile oils of nine populations of six species from Albania, namely Artemisia absinthium, Calamintha nepeta, Hypericum perforatum, Sideritis raeseri subsp. raeseri, Origanum vulgare subsp. hirtum from two wild .populations, and Salvia officinalis (sage) from two wild and one cultivated population,. The essential oils were obtained by hydrodistillation and their analyses were performed by GC-MS. The major constituents were: A. absinthium: neryl isovalerate (19.5%), geranyl isobutanoate (16.4%) and carvacrol (8.8%); C. nepeta: pulegone (31.7%), spathulenol (20.0%) and isomenthone (12.7%); H. perforatum: caryophyllene oxide (31.0%), δ-selinene (10.5%) and carvacrol (10.4%); O. vulgare: carvacrol (81.0, 78.6%), y-terpinene (5.5, 7.1%) and p-cymene (4.9, 4.1%) for 0. vulgare originating from Tepelena. and.Vlora, respectively; S. raeseri: carvacrol (36.7%), caryophyllene oxide (17.8%), p-caryphyllene (8.7%), spathulenol (7.7%) and myrtenol (6.4%); S. officinalis: camphor (40.2, 47.8, 45.9%), c-thujone .(19.2, 22.2, 13.7%), eucalyptol (5.4, 2.6, 6.0%), camphene (5.8, 6.1, 3.9, %), borneol (2.1, 2.9, 5.7%) and bornyl acetate (3.3, 1.4, 5.6%) for samples originating from Tepelena, Tirana and Vlora, respectively. The essential oils were also tested for their free radical scavenging activity using the following in vitro assays: i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), and ii) inhibition of linoleic acid-peroxidation with 2,2'-azobis- 2-methyl-propanimidamide, dihydrochloride (AAPH). Finally, their inhibitory activity toward soybean lipoxygenase was evaluated, using linoleic acid as substrate.The essential oil of 0. vulgare (OV-VL) presented the highest interaction with the stable radical DPPH (76.5%), followed by that of A. absinthium (54.7%). O. vulgare (OV-TP) and A. absinthium showed high anti-lipid peroxidation activity, 97.5% and 96.5%, respectively, higher than that of the reference compound trolox (73.0%). Only the tested sample of O. vulgare (OV-VL) significantly inhibited soybean lipoxygenase (54.2%).

Lignans and indole alkaloids from the seeds of Centaurea vlachorum Hartvig (Asteraceae), growing wild in Albania and their biological activity.

The present phytochemical investigation of the seeds of Centaurea vlachorum led to the isolation and characterisation of four compounds including two indole alkaloids N-(p-coumaroyl) serotonin (1) and moschamine (2) and two dibenzylbutyrolactone lignans matairesinol (3) and arctiin (4). This is the first report on the isolation of non-volatile secondary metabolites from C. vlachorum. The chemataxonomic significance of these compounds was summarised. Moreover, the isolated compounds were tested for their free radical scavenging activity using the following in vitro assays: (i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), (ii) inhibition of linoleic acid peroxidation with the dihydrochloric acid of 2,2-Azobis-2-amidinopropane (AAPH). Finally, their inhibitory activity towards soybean lipoxygenase was evaluated, using linoleic acid as substrate.