Papers by Maria Halabalaki
Metabolites
Oleuropein (OE) is a secoiridoid glycoside occurring mostly in the Oleaceae family and presenting... more Oleuropein (OE) is a secoiridoid glycoside occurring mostly in the Oleaceae family and presenting several pharmacological properties, including hypolipidemic and antioxidant properties. Based on these, several dietary supplements containing olive leaf extracts enriched with OE are commercially available in many countries. The current study aimed to examine the effect of supplementation with such an extract on the serum and urine metabolome of young healthy male athletes. For this purpose, applying a randomized, balanced, double-blind study, nine young, healthy males (physical education students) received either a commercially prepared extract or placebo for one week, followed by a two-week washout period; then, they were subsequently dosed with the alternate scheme (crossover design). Urine and serum samples were analyzed using UHPLC-HRMS, followed by evaluation with several multivariate methods of data analysis. The data were interpreted using a multilevel metabolomic approach (mul...
Molecules, 2020
In recent years, isotopic analysis has been proven a valuable tool for the determination of the o... more In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the 18O and 13C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ18O isotopic values range from 19.2 ‰ to 25.2 ‰ and the δ13C values range from −32.7 ‰ to −28.3 ‰. These differences between the olive oils’ isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the 13C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.
European Heart Journal, 2021
The main bioactive compounds of olive products, oleuropein (OL), hydroxytyrosol (HT), oleocanthal... more The main bioactive compounds of olive products, oleuropein (OL), hydroxytyrosol (HT), oleocanthal (OC) and oleanolic Acid (OA) exert multiple benefits in cardiovascular diseases. However, their potential cardioprotective effect after chronic administration at a nutritional dose has not been studied in metabolic syndrome (MS). We evaluated and compared possible cardioprotective effects of chronic oral treatment with OL, HT, OC and OA in a mouse model of diet-induced MS. We initially explored if OL, HT, OC, OA exert cardioprotection against ischemia-reperfusion (I/R) injury after chronic administration in healthy animals. The nutritional dose was selected as the equivalent to the estimated daily phenolic intake in humans following the Mediterranean diet. C57Bl6 mice were randomized into 5 groups and treated daily for 6 weeks as followed: i) DMSO 5%, ii) OL (20.6 mg/kg), iii) HT (5.9 mg/kg), iv) OC (11.6 mg/kg), v) OA (17.4 mg/kg). All animals were then subjected to I/R (30/120 min) an...
Journal of Medicinal Plants Studies, 2021
A recent ethnobotanical study collected traditional knowledge on treatments and plants of tribes ... more A recent ethnobotanical study collected traditional knowledge on treatments and plants of tribes in Uige, Northern Angola, a region suffering from infant malnutrition and mortality. Aqueous extracts of twelve different plant species (leaves or stems) collected were prepared and subject of chemical profiling. The impact of twelve plant extracts on mammary epithelial cell differentiation was tested in vitro by dose-dependent treatment of HC11 cells. Leaf extracts of Carica papaya and Morinda lucida induced HC11 differentiation and elevated the mRNA expression of the milk protein β-casein, while the remaining ten inhibited the HC11 mammary differentiation.The majority of the plant extracts did not induce in vitro mammary epithelial cell differentiation assuming that the plants might rather exert their effects on prolactin levels or on other mechanism influencing the mammary gland health. Phenolic acids and flavonoids were identified as the predominant and potentially active chemical cl...
Planta Medica, 2021
Chios mastic gum is the resinous secretion obtained from the barks of the shrub Pistacia lentiscu... more Chios mastic gum is the resinous secretion obtained from the barks of the shrub Pistacia lentiscus var. Chia, which is endemic to the Greek island of Chios. Since antiquity, Chios mastic gum has found several uses as a phytotherapeutic remedy, primarily for the treatment of gastrointestinal disorders while recently, Chios mastic gum was also recognized by EMA as an herbal medicinal product with specific indications. Chios mastic gumʼs biological properties are attributed to triterpenes which comprise the major chemical group (approx. 70%) and notably isomasticadienonic acid and masticadienonic acid. However, due to their structural characteristics, the isolation thereof in high yield and purity is challenging and since they are not commercially available, pharmacological studies aiming to assess their biological properties are limited. In the present work, masticʼs phytochemical investigation by UPLC-HRMS is followed by the isolation and characterization of isomasticadienonic acid a...
Food Chemistry, 2021
Ruby chocolate was introduced in 2017 as the fourth type of chocolate, in addition to white, milk... more Ruby chocolate was introduced in 2017 as the fourth type of chocolate, in addition to white, milk and dark chocolate. However, until now not much is reported about its phytochemical composition. Therefore, we analyzed ruby chocolate by UPLC-HRMS, together with the three other types of chocolate. Feature-based molecular networking was carried out to aid in the identification, while a set of 51 reference compounds were analyzed simultaneously for targeted quantification. In this way, a total of 54 compounds could be (tentatively) identified in the chocolates, of which 43 were found in the ruby chocolate. Moreover, 19 compounds were quantified, of which 13 in the ruby chocolate. The compounds include flavan-3-ols, proanthocyanidins and methylxanthines, but also biogenic amines and alkaloids. In general, ruby chocolate contained lower levels of these constituents compared to dark chocolate. However, A-type proanthocyanidins were found to be characteristic for the ruby chocolate.
Processes, 2021
Chios Mastic Gum (CMG) and Chios Mastic Oil (CMO) are two unique products of the tree Pistacia le... more Chios Mastic Gum (CMG) and Chios Mastic Oil (CMO) are two unique products of the tree Pistacia lentiscus var. Chia, cultivated exclusively on the Greek island of Chios. In the present study, the method proposed by the European Pharmacopoeia for mastic identification was employed using HPTLC together with an in-house method. A GC-MS methodology was also developed for the chemical characterization of CMOs. α-Pinene and β-myrcene were found in abundance in the fresh oils; however, in the oil of the aged collection, oxygenated monoterpenes and benzenoids such as verbenone, pinocarveol, and α-campholenal were found at the highest rates. Additionally, the antimicrobial activity of Chios Mastic Gums (CMGs) with their respective Chios Mastic Oils (CMOs) was evaluated, with growth tests against the fungi Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, Mucor circinelloides, and Rhizopus oryzae, and the bacteria Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, wit...
Phytochemistry, 2020
MicroRNAs (miRNAs) play a pivotal role in plant development. The expression patterns of the miRNA... more MicroRNAs (miRNAs) play a pivotal role in plant development. The expression patterns of the miRNA genes significantly influence their regulatory activities. By utilizing small RNA (sRNA) high-throughput sequencing (HTS) data, the miRNA expression patterns were investigated in four organs (flowers, leaves, roots and seedlings) of Arabidopsis. Based on a set of criteria, dozens of organ-specific miRNAs were discovered. A dominant portion of the organ-specific miRNAs identified from the ARGONAUTE 4-enriched sRNA HTS libraries were highly expressed in flowers. Additionally, the expression of the precursors of the organ-specific miRNAs was analyzed. Degradome sequencing data-based approach was employed to identify the targets of the organ-specific miRNAs. The miRNA-target interactions were used for network construction. Subnetwork analysis unraveled some novel regulatory cascades, such as the feedback regulation mediated by miR161, the potential self-regulation of the genes miR172, miR396, miR398 and miR860, and the miR863-guided cleavage of the SERRATE transcript. Our bioinformatics survey expanded the organ-specific miRNA-target list in Arabidopsis, and could deepen the biological view of the miRNA expression and their regulatory roles.
Scientific Reports, 2017
Proteasome inhibitors, e.g. Bortezomib (BTZ) and Carfilzomib (CFZ), have demonstrated clinical ef... more Proteasome inhibitors, e.g. Bortezomib (BTZ) and Carfilzomib (CFZ), have demonstrated clinical efficacy against haematological cancers. Interestingly, several adverse effects are less common, compared to BTZ, in patients treated with CFZ. As the molecular details of these observations remain not well understood we assayed the pathophysiological effects of CFZ vs. BTZ in the Drosophila experimental model. Mass Spectrometry analyses showed that neither CFZ nor BTZ are hydrolysed in flies’ tissues, while at doses inducing similar inhibition of the rate limiting for protein breakdown chymotrypsin-like (CT-L) proteasomal activity, CFZ treatment resulted in less intense increase of oxidative stress or activation of antioxidant and proteostatic modules. Also, despite comparable cardiotoxicity likely due to disrupted mitochondrial function, CFZ did not affect developmental processes, showed minimal neuromuscular defects and reduced to a lesser extent flies’ healthspan. Studies in flies, hum...
PLOS ONE, 2018
In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents co... more In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations �0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08-5.00 mg ml-1) and S. sclarea (MIC: 0.08-2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1 , respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15-2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections.
Journal of Pharmaceutical and Biomedical Analysis, 2019
Indirubins represent a group of natural and synthetic products with bio-activities against numero... more Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6-bromo indirubin-3′-oxime (6BIO) possesses increased selectivity against GSK-3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 µm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half-life of 1 h, and low plasma bioavailability.
Redox biology, 2018
Natural products are characterized by extreme structural diversity and thus they offer a unique s... more Natural products are characterized by extreme structural diversity and thus they offer a unique source for the identification of novel anti-tumor agents. Herein, we report that the herbal substance acteoside being isolated by advanced phytochemical methods from Lippia citriodora leaves showed enhanced cytotoxicity against metastatic tumor cells; acted in synergy with various cytotoxic agents and it sensitized chemoresistant cancer cells. Acteoside was not toxic in physiological cellular contexts, while it increased oxidative load, affected the activity of proteostatic modules and suppressed matrix metalloproteinases in tumor cell lines. Intraperitoneal or oral (via drinking water) administration of acteoside in a melanoma mouse model upregulated antioxidant responses in the tumors; yet, only intraperitoneal delivery suppressed tumor growth and induced anti-tumor-reactive immune responses. Mass-spectrometry identification/quantitation analyses revealed that intraperitoneal delivery o...
Antioxidants & Redox Signaling, 2017
Aims: Organismal aging can be delayed by mutations that either activate stress responses or reduc... more Aims: Organismal aging can be delayed by mutations that either activate stress responses or reduce the nutrientsensing pathway signaling; thus, by using Drosophila melanogaster as an in vivo experimental screening platform, we searched for compounds that modulate these pathways. Results: We noted that oral administration of the glycogen synthase kinase 3 (Gsk-3) inhibitor 6-bromoindirubin-3¢-oxime (6BIO) in Drosophila flies extended healthy life span. 6BIO is not metabolized in fly tissues, modulated bioenergetic pathways, decreased lipid and glucose tissue load, activated antioxidant and proteostatic modules, and enhanced resistance to stressors. Mechanistically, we found that the effects on the stress-responsive pathways were largely dependent on the activity of the transcription factor nuclear factor erythroid 2-related factor (Nrf-2). Genetic inhibition of Gsk-3 largely phenocopied the 6BIO-mediated effects, while high levels of Gsk-3 expression and/or kinase activity suppressed proteostatic modules and reduced flies' longevity; these effects were partially rescued by 6BIO. Also, 6BIO was found to partially reduce the 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) activity, a major effector of the insulin/insulin-like growth factor-1 cell signaling pathways. Innovation: 6BIO exerts the unique property of increasing stress tolerance and in parallel partially suppressing the nutrient-sensing pathway signaling. Conclusion: Our findings suggest that the 6BIO scaffold can be used for the development of novel antiaging compounds.
ChemistrySelect, 2016
The discovery of new active natural products is hampered by laborious purification processes that... more The discovery of new active natural products is hampered by laborious purification processes that often end up to the re‐isolation of known compounds. We demonstrate here that, spectral data reflecting concentration fluctuations of components can correlate statistically with measurable dose‐dependent properties on the basis of a Heterocovariance approach deconvoluting the active components structure. Variance of extract constituents was achieved through statistically meaningful collections of plants from different families, genus, and species. This fluctuation was also obtained through the fractionation of a single plant extract by separation techniques. The NMR and HRMS spectra of the extracts and fractions were recorded, as well as their ability to inhibit tyrosinase or 5‐lipoxygenase enzymes. Biological activity was statistically correlated with spectral data deciphering the active compounds through the Heterocovariance approach prior to any purification.
Pharmaceutical biology, Jan 7, 2015
Eythrina excelsa Baker (Fabaceae) is a medicinal plant used to treat various ailments including t... more Eythrina excelsa Baker (Fabaceae) is a medicinal plant used to treat various ailments including those of the female genital tract. The objective of this study is to investigate the estrogenic and cytotoxic effects of the ethanol extract of the stem bark of E. excelsa. Erythrina excelsa was evaluated in vitro using the yeast estrogen screen (YES). The extract was then tested in a 3-day uterotrophic assay on ovariectomised Wistar rats at doses of 50 and 100 mg/kg BW/d. Cytotoxic effects were assessed on breast (MCF-7) and colon (HT-29) cancer cell lines using the MTT cell viability assay. Additionally, a LC-PDA-ESI (+)-HRMS and HRMS/MS method was developed and applied for the identification of representative secondary metabolites scaffolds in the extract. In the YES, the extract stimulated the transactivation of the estrogen receptor in a dose-dependent manner with an EC50 value of 1.8 μg/mL. In rats, E. excelsa increased uterine wet weight, uterine epithelial height, and the mRNA exp...
International Scholarly Research Notices, 2014
Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was s... more Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its sec...
Revista Brasileira de Farmacognosia, 2013
It is now widely-recognized that the view that herbal remedies have no adverse effects and/or tox... more It is now widely-recognized that the view that herbal remedies have no adverse effects and/or toxicity is incorrect; some traditionally-used plants can present toxicity. The well-established popular use of Ageratum conyzoides has led to its inclusion in a category of medicinal crude drugs created by the Brazilian Health Surveillance Agency. Ageratum belongs to the Eupatorieae tribe, Asteraceae, and is described as containing toxic pyrrolizidine alkaloids. Aqueous extracts of Ageratum conyzoides L. harvested in Brazil (commercial, fl owering and non-fl owering samples) were prepared according to the prescribed method and analyzed by HPLC-HRMS. The pyrrolizidine alkaloids lycopsamine, dihydrolycopsamine, and acetyl-lycopsamine and their N-oxides, were detected in the analyzed extracts, lycopsamine and its N-oxide being known hepatotoxins and tumorigens. Together with the pyrrolizidine alkaloids identifi ed by HPLC-HRMS, thirteen phenolic compounds were identifi ed, notably, methoxylated fl avonoids and chromenes. Toxicological studies on A. conyzoides are necessary, as is monitoring of its clinical use. To date, there are no established safety guidelines on pyrrolizidine alkaloids-containing plants, and their use in Brazil.
Molecules, 2014
Micromelum falcatum, a medicinal plant of the Rutaceae family, has been used in the Traditional C... more Micromelum falcatum, a medicinal plant of the Rutaceae family, has been used in the Traditional Chinese Medicine (TCM) mainly against colds and rheumatoid arthritis. Despite its traditional use the association of its constituents with possible anti-inflammatory activity has not been explored. During this study, a rapid UPLC-ESI(+)-HRMS method was developed for the profiling of M. falcatum leave extracts and the targeted isolation of coumarin constituents. Based on chromatographic, spectroscopic and spectrometric features several 7-oxygenated coumarin derivatives were detected. After targeted isolation, eight coumarins, among them three new natural products, namely microfalcrin, microcoumaririn and micromelosidester, were purified using semi-preparative HPLC and unambiguously identified by 1 and 2D NMR. Furthermore, important spectrometric characteristics were revealed based on the HRMS and HRMS/MS spectra of the isolated 7-oxygenated coumarins facilitating their identification in complex mixtures. Finally, the anti-inflammatory properties of the extracts and representative compounds were evaluated
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Papers by Maria Halabalaki