Determination of the Total Phenolics Content and Antioxidant Activity of Extracts from Parts of Plants from the Greek Island of Crete.

Oxidative damages are responsible for many adverse health effects and food deterioration. The use of antioxidant substances is well renowned, and as such, much emphasis is placed on their use. Since synthetic antioxidants exhibit potential adverse effects, plant-derived antioxidants are a preferable solution. Despite the myriads of plants that exist and the fact that numerous studies have been carried out so far, there are many species that have not been examined so far. Many plants under research exist in Greece. Trying to fill this research gap, the total phenolics content and antioxidant activity of seventy methanolic extracts from parts of Greek plants were evaluated. The total phenolics content was measured by the Folin-Ciocalteau assay. Their antioxidant capacity was calculated by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging test, the Rancimat method based on conductometric measurements, and the thermoanalytical method DSC (Differential Scanning Calorimetry). The tested samples were obtained from several parts of fifty-seven Greek plant species belonging to twenty-three different families. Both a remarkably high phenolic content (with gallic acid equivalents varying between 311.6 and 735.5 mg/g of extract) and radical scavenging activity (IC50 values ranged from 7.2 to 39.0 µg/mL) were found in the extract of the aerial parts of Cistus species (C. creticus subsp. creticus, C. creticus subsp. eriocephalus, C. monspeliensis, C. parviflorus and C. salviifolius), Cytinus taxa (C. hypocistis subsp. hypocistis, C. hypocistis subsp. orientalis and C. ruber), and Sarcopoterium spinosum. Furthermore, the sample of Cytinus ruber showed the highest protection factor (PF = 1.276) regarding the Rancimat method, which was similar to that of butylated hydroxytoluene (BHT) (PF = 1.320). The results indicated that these plants are rich in antioxidant compounds, potentiating their use either as food additives to enhance the antioxidant properties of food products, or protect them from oxidation, or as sources for the preparation of food supplements with antioxidant properties.

A GC-MS and LC-HRMS perspective on the chemotaxonomic investigation of the natural hybrid Origanum × lirium and its parents, O. vulgare subsp. hirtum and O. scabrum.

INTRODUCTION: The genus Origanum L. (Lamiaceae) is widespread in the Mediterranean region. However, approximately 75% of the species are only encountered in the eastern part. Out of these, a total of nine species (11 taxa) and three natural hybrids occur in Greece. Nevertheless, so far, there is no consensus regarding their precise botanical classification in the literature. In fact, the taxon Origanum × lirium has been proposed both as a separate species as well as natural hybrid between Origanum vulgare subsp. hirtum and Origanum scabrum. OBJECTIVES: In this scope, the aim of the current study is to shed light on the matter through the investigation of the chemical composition of both the essential oils and the polar extracts of the mentioned taxa, collected from different geographical regions of Greece. RESULTS: As it was demonstrated by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) data, and highlighted by our comparative analysis, it can be stipulated that Origanum × lirium shares its chemotype to a large extent with its parent species concerning both volatile and polar constituents. Additionally, geographical origin conditions stood out as a key factor influencing their chemical composition. CONCLUSION: Altogether, the present work provides useful information on the chemical composition of the taxa under investigation, while our findings support the opinion that Origanum × lirium should be considered not as a separate species, but rather as a hybrid on the way to becoming a species.

A Bio-Guided Screening for Antioxidant, Anti-Inflammatory and Hypolipidemic Potential Supported by Non-Targeted Metabolomic Analysis of Crepis spp.

This study was designed to evaluate the chemical fingerprints and the antioxidant, anti-inflammatory and hypolipidemic activity of selected Crepis species collected in Greece, namely, C. commutata, C. dioscoridis, C. foetida, C. heldreichiana, C. incana, C. rubra, and Phitosia crocifolia (formerly known as Crepis crocifolia). For the phytochemical analyses, sample measurements were carried out by using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS). Τhe extracts were evaluated both in vitro (radical scavenging activity: DPPH assay and total phenolic content: Folin-Ciocalteu) and in vivo (paw edema reduction and hypolipidemic activity: experimental mouse protocols). Among the tested extracts, C. incana presented the highest gallic acid equivalents (GAE) (0.0834 mg/mL) and the highest antioxidant activity (IC50 = 0.07 mg/mL) in vitro, as well as the highest anti-inflammatory activity with 32% edema reduction in vivo. Moreover, in the hypolipidemic protocol, the same extract increased plasma total antioxidant capacity (TAC) by 48.7%, and decreased cholesterol (41.3%) as well as triglycerides (37.2%). According to fractionation of the extract and the phytochemical results, this biological effect may be associated with the rich phenolic composition; caffeoyl tartaric acid derivatives (cichoric and caftaric acid) are regarded as the most prominent bioactive specialized metabolites. The present study contributes to the knowledge regarding the phytochemical and pharmacological profile of Crepis spp.

Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin.

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2- breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

Phytochemical Study and In Vitro Screening Focusing on the Anti-Aging Features of Various Plants of the Greek Flora.

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging.

Magnoflorine-Isolation and the Anticancer Potential against NCI-H1299 Lung, MDA-MB-468 Breast, T98G Glioma, and TE671 Rhabdomyosarcoma Cancer Cells.

Magnoflorine (MGN) is a quaternary aporphine alkaloid that exhibits numerous therapeutic properties, including neuropsychopharmacological, anti-anxiety, immunomodulatory, anti-inflammatory, antioxidant, or antifungal activities. The aim of the present study was an investigation of the influence of MGN on viability, proliferation, induction of apoptosis, and cell cycle arrest in NCI-H1299 lung, MDA-MB-468 breast, T98G glioma, and TE671 rhabdomyosarcoma cancer cells. MGN was isolated from the roots of Berberis cretica L. by counter-current partition chromatography (CPC). Cell viability and proliferation assessments were performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and 5-bromo-2'-deoxyuridine (BrDU) assays, respectively. The induction of apoptosis and cell cycle progression was measured using fluorescence-activated cell sorting analysis. MGN in high doses inhibits proliferation, induces apoptosis, and inhibits cell cycle in S/G2 phases in a dose-dependent manner. MGN seems to be a promising anti-cancer compound in therapy of some types of lung, breast, glioma, and rhabdomyosarcoma cancers, for which current standard therapies are limited or have severe strong side effects.

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. AIM OF THE STUDY: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. MATERIALS AND METHODS: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. RESULTS: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 µg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 µM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 µM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. CONCLUSIONS: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones.

The purpose of this study was to evaluate the response of estrogen target cells to a series of isoflavone glucosides and aglycones from Genista halacsyi Heldr. The methanolic extract of aerial parts of this plant was processed using fast centrifugal partition chromatography, resulting in isolation of four archetypal isoflavones (genistein, daidzein, isoprunetin, 8-C-ß-D-glucopyranosyl-genistein) and ten derivatives thereof. 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein were among the most abundant constituents of the isolate. All fourteen, except genistein, displayed low binding affinity for estrogen receptors (ER). Models of binding to ERα could account for the low binding affinity of monoglucosides. Genistein and its glucosides displayed full efficacy in inducing alkaline phosphatase (AlkP) in Ishikawa cells, proliferation of MCF-7 cells and ER-dependent gene expression in reporter cells at low concentrations (around 0.3 µM). ICI182,780 fully antagonized these effects. The AlkP-inducing efficacy of the fourteen isoflavonoids was more strongly correlated with their transcriptional efficacy through ERα. O-monoglucosides displayed higher area under the dose-response curve (AUC) of AlkP response relative to the AUC of ERα-transcriptional response compared to the respective aglycones. In addition, 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein displayed estradiol-like efficacy in promoting differentiation of MC3T3-E1 cells to osteoblasts, while genistein was not convincingly effective in this respect. Moreover, 7,4΄-di-O-ß-D-glucopyranosyl-genistein suppressed lipopolysaccharide-induced tumor necrosis factor mRNA expression in RAW 264.7 cells, while 7-O-ß-D-glucopyranosyl-genistein was not convincingly effective and genistein was ineffective. However, genistein and its O-glucosides were ineffective in inhibiting differentiation of RAW 264.7 cells to osteoclasts and in protecting glutamate-challenged HT22 hippocampal neurons from oxidative stress-induced cell death. These findings suggest that 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein display higher estrogen-like and/or anti-inflammatory activity compared to the aglycone. The possibility of using preparations rich in O-ß-D-glucopyranosides of genistein to substitute for low-dose estrogen in formulations for menopausal symptoms is discussed.

Phytochemical Composition of the Decoctions of Greek Edible Greens (Chórta) and Evaluation of Antioxidant and Cytotoxic Properties.

Wild or semi-wild edible greens (chórta) are an integral part of the traditional Greek Mediterranean diet due to their nutritional value, containing various phytonutrients beneficial to human health. Water-based decoctions of chórta are widely consumed in Greek alternative medicine as health promoting agents. This study examined the chemical profile of the decoctions of eight edible plants, Cichorium intybus, C. endivia, C. spinosum, Crepis sancta, Sonchus asper, Carthamus lanatus, Centaurea raphanina, and Amaranthus blitum, by UPLC-ESI-HRMS and HRMS/MS analysis, to determine possibly bioactive constituents. The profiles of the plants from the Asteraceae family are dominated by the presence of phenolic acids and flavonoid derivatives, whereas the A. blitum decoction is rich in triterpene saponins. Interestingly, the Centaurea raphanina decoction was found to be extremely rich in flavanones, particularly in the aglycone pinocembrin. Further phytochemical investigation and fractionation of this extract resulted in the isolation and identification of five compounds: phlorin (1), syringin (2), pinocembrin (3), pinocembroside (4), and pinocembrin-7-O-neohesperidoside (5). The extracts were also tested for their antioxidant and differential cytotoxic activity against tumor cells. C. raphanina was found to be differentially toxic against metastatic tumor cells. In conclusion, we found that Greek edible greens are a rich source of bioactive secondary metabolites and their consumption could contribute to the maintenance of overall health.

Phytotoxic triterpene saponins from Bellis longifolia, an endemic plant of Crete.

In a study of 62 plant species of the Cretan flora for their phytotoxic activity, plants were extracted successively with CH2Cl2, MeOH and H2O. Phytotoxicity evaluation of the 240 extracts was performed against Lactuca sativa L. and Agrostis stolonifera L.. The MeOH extract of Bellis longifolia was the most phytotoxic. Bioassay-guided fractionation revealed that a fraction consisting mainly of saponins was the most effective. Separation of the saponins was performed using initially a step-gradient Centrifugal Partition Chromatography (CPC). Investigation of the active fraction led to the isolation and structure elucidation of three previously undescribed triterpene saponins, 3-O-ß-D-fucopyranosyl polygalacic acid, 28-O-α-L-rhamnopyranosyl-(1 â†’ 2)-ß-D-fucopyranosyl polygalacic acid and 3-O-ß-D-fucopyranosyl-2α,3ß,23-trihydroxyolean-12-en-28-oic acid, which were present as the main phytotoxic compounds of the methanol extract. Two triterpenes, polygalacic acid and bellisonic acid and four kaempferol glucosides, as well as chlorogenic acid were also isolated. 3-O-ß-D-fucopyranosyl polygalacic acid and 3-O-ß-D-fucopyranosyl-2α,3ß,23-trihydroxyolean-12-en-28-oic acid had phytotoxic activity similar to some commercial herbicides (IC50 values of ca. 25 µM) against duckweed (Lemna paucicostata).

Influence of extraction procedures on phenolic content and antioxidant activity of Cretan barberry herb.

The main goal of present study was the development, optimization and application of different extraction protocols, especially those employing green technologies, in order to obtain from Berberis cretica extracts with high antioxidant capacity. For this purpose, the applied methods: maceration, ASE and SFE coupled with ASE were incorporated. The antioxidant assessment was carried out using DPPH and total phenolic content (Folin-Ciocalteu) assays. Major constituents were elucidated using HPLC-DAD and UHPLC-HRMS/MS (hybrid IT-Orbital trap spectrometer) equipped with an ESI probe. The chromatographic and spectral data revealed the presence of several simple phenolic acids, derivatives of both caffeic and benzoic acids, and flavonoids in the produced extracts. It was clearly evidenced that the extraction method and solvents used affected both the activity and the chemical content of the results, significantly. The most beneficial conditions were calculated for methanol and water:ethanol (50:50) extracts derived from the combination of SFE and ASE methodologies. Obtained results classify Cretan barberry as a strong antioxidant agent.

Polygonophenone, the first MEM-substituted natural product, from Polygonum maritimum.

An unprecedented natural acetophenone, polygonophenone (1), and a new resorcinol, polygonocinol (2), were isolated from the dichloromethane and methanol extracts of Polygonum maritimum and identified as 2-hydroxy-4-[(2-methoxyethoxy)methoxy] acetophenone and 2-methyl-5-nonadecylresorcinol, respectively. In addition, 11 known compounds were identified, namely, the sesquiterpenoid (+)-8-hydroxycalamene, four ferulic acid esters (tetracosyl, hexacosyl, octacosyl, and triacontyl ferulate), the arylpropane broussonin B, the flavonoids quercetin, quercitrin, and (+)-catechin, the hydroquinone glucoside isotachioside, and beta-sitosterol. The structures of the new compounds were elucidated on the basis of their NMR and MS data. It is noteworthy that polygonophenone (1) is the first naturally methoxyethoxymethyl (MEM)-substituted natural product, and its isolation gives support for the use of MEM protection in biomimetic synthetic schemes.

Antileishmanial activity of natural diterpenes from Cistus sp. and semisynthetic derivatives thereof.

Eleven cis-clerodane diterpenes, seven labdane type diterpenes and one triterpene isolated from Cistus monspeliensis and the resin "Ladano" of Cistus creticus subsp. creticus were evaluated against Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis. In addition, eleven semisynthetic manoyl oxide, seventeen labdane type derivatives and a triterpene were also evaluated for their antileishmanial activity. 18-Acetoxy-cis-clerod-3-en-15-ol, 15,18-diacetoxy-cis-clerod-3-ene and 13-(E)-8a-hydroxylabd-13-en-15-ol 2-chloroethylcarbamate exhibited the most potent and selective leishmanicidal activity with IC50 values of 3.3 microg/ml, 3.4 microg/ml and 3.5 microg/ml, respectively.

Characterization of the essential oil volatiles of Satureja thymbra and Satureja parnassica: influence of harvesting time and antimicrobial activity.

Seasonal variation in chemical composition of essential oils obtained from Satureja parnassica subsp. parnassica Heldr. & Sart ex Boiss. and Satureja thymbra L. was determined by GC and GC-MS analysis. The in vitro antibacterial activities of these essential oils were evaluated at two different initial levels against two common foodborne bacteria. Their minimum inhibitory concentration (MIC) and noninhibitory concentration (NIC) values were determined via an innovative automated technique, which was applied on a Malthus apparatus combining the conductance measurements with the common dilution method. The essential oil concentration fluctuated greatly during the period examined and established that the Satureja oils obtained during the flowering period were the most potent, exhibiting the lowest MIC values and retaining remarkable bactericidal properties. Thus, they represent an inexpensive source of natural antibacterial substances that exhibit potential for use in food systems to prevent the growth of foodborne bacteria and to extend the shelf life of the processed food.

Essential oils of Satureja, Origanum, and Thymus species: chemical composition and antibacterial activities against foodborne pathogens.

The chemical composition of the essential oils obtained from the species restricted to Greece and the eastern Mediterranean region, Satureja spinosa L. and Thymus longicaulis L.; species endemic to central and south Greece, Satureja parnassica ssp. parnassica Heldr. and Sart ex Boiss.; species endemic to the island of Crete, Origanum dictamnus L.; and species widely distributed in the Mediterranean region, Satureja thymbra L. and Origanum vulgare L. subsp. hirtum, were determined by gas chromatography (GC) and GC/mass spectrometry (MS) analysis. The in vitro antibacterial activities of the essential oils were evaluated against a panel of five foodborne bacteria (Escherichia coli 0157:H7 NCTC 12900, Salmonella enteritidis PT4, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ScottA, and Bacillus cereus FSS 134). The analytical data indicated that various monoterpene hydrocarbons and phenolic monoterpenes constitute the major components of the oils, but their concentrations varied greatly among the oils examined. The antibacterial assay results showed that 5 muL doses of the essential oils extracted from the endemic Satureja species in Greece possess remarkable bactericidal properties, which are clearly superior as compared to those of Origanum and Thymus species essential oils. Therefore, they represent an inexpensive source of natural mixtures of antibacterial compounds that exhibit potentials for use in food systems to prevent the growth of foodborne bacteria and extend the shelf life of the processed food.

cis-Clerodane type diterpenes from Cistus monspeliensis.

The aerial parts of Cistus monspeliensis yielded 11 cis-clerodane type diterpenes. Five of these, 3, 4, 8,9, and 10, are new compounds. Their structures have been elucidated on the basis of their spectral data (FABMS, 1D and 2D NMR).

Paeonicluside, a new salicylic glycoside from the Greek endemic species Paeonia clusii.

A new glycoside of salicylic aldehyde, paeonicluside, was isolated from the roots of the Greek endemic species Paeonia clusii subsp. clusii and identified as alpha-L-arabinopyranosyl-(1-->6)-O-beta-D-glucopyranoside of salicylic aldehyde (1). In addition, one characteristic monoterpene and two monoterpene glycosides were identified as paeoniflorigenone, paeoniflorin and benzoyl paeoniflorin, respectively. The structure of 1 was elucidated on the basis of its spectroscopic data and chemical correlation. It is the first time that a derivative of salicylic aldehyde is isolated from the well-studied Paeonia genus.

Volatiles with antimicrobial activity from the roots of Greek Paeonia taxa.

The traditional uses of the roots of Paeonia taxa are described. The volatile constituents, obtained by steam distillation of the roots of Paeonia clusii Stern subsp. clusii, Paeonia mascula L. subsp. hellenica and Paeonia parnassica, were studied by GC/MS. Salicylaldehyde, paeonol, methyl salicylate and benzoic acid were found to be the major components among the twelve constituents identified. The evaluation of the in vitro antimicrobial activity of the volatile compounds against six bacteria and three fungi is also reported.