Proline-based deep eutectic solvents as greener alternative for obtaining polyphenol rich extracts of Satureja kitaibelii

INTRODUCTION: Aerial parts of Satureja kitaibelii Wierzb. ex Heuff. (Lamiaceae), in Serbia known as Rtanj’s tea, are traditionally used to treat various respiratory, urinary and other health disorders. Extracts of this herb exhibit a significant bioactivity as well37. Using deep eutectic solvents (DESs) for extraction of certain phenolic compounds is in line with the principles of green chemistry38. However, the toxicity of DESs must be considered39. OBJECTIVES: The main objectives of this work are to assess the polyphenol-extracting ability of proline (Pro) and sugar/sugar alcohol based natural DESs from commercially available Rtanj’s tea, as well as to evaluate cytotoxicity of these solvents against AsPC- 1 cells. METHOD / DESIGN: Accurately weighed Pro and sugar/sugar alcohol, were dissolved in water, frozen, and freeze-dried. The obtained seven DESs (i.e. Pro with: glucose 1:1 and 5:3, fructose 1:1 and 5:3, sorbitol 1:2, and sucrose 2:1 and 3:2) were mixed with water (30%, m/m). Polyphenol extraction was examined using commercial sample of Rtanj’s tea (manufacturer Bojan Radosavljević, Boljevac). Powdered herb (particle diameter 100-200 μm) was extracted by sonication during 30 min at room temperature with the obtained aqueous DESs (herb-to-solvent ratio 1:20), as well as with water, absolute ethanol, or 50% (v/v) ethanol under the same conditions. The qualitative analysis of extracts was conducted by both HPLC and LC-MS. The content of the identified marker compounds in the extracts, i.e. rosmarinic acid (RA) and clinopodic acid O (CAO), was determined by external calibration using RA as the standard compound. Cytotoxicity of the aqueous DESs against human pancreatic adenocarcinoma cells AsPC-1 was tested at four concentration levels (5-25%), after 48 h of incubation and propidium iodide staining. The analysis on Guava® easyCyte 12HT Benchtop flow microcapillary cytometer, was performed afterwards, using InCyte® software package. RESULTS: Upon freeze-drying, mixtures had glassy appearance and transformed into liquids after mild heating. All obtained DESs were highly viscous, and therefore mixed with water. Qualitative LC-MS analysis of 50% ethanol extract revealed the presence of phenolic acids, flavonoids, and jasmonic acid derivatives. Among phenolic acids, the dominant compounds were caffeic acid oligomers RA and CAO. Among conventional solvents, 50% ethanol was better extracting agent than absolute ethanol or water for both RA (88.2 μg/mL) and CAO (116.8 μg/mL). Water extract was also abundant with CAO (106.7 μg/mL), but contained moderate amount of RA (21.7 μg/mL). It is noteworthy to mention that the extraction with ethanol resulted in very low yield of both phenolics, with CAO concentration, being even below detection limit in the absolute ethanol extract. Concentration of RA in the tested DES extracts was higher than corresponding one in the water extract, but lower than in the 50% ethanol extract, and varied in the range from 61.6 μg/mL (in Pro-fructose 1:1 extract) to 85.6 μg/mL (Pro-glucose 1:1 extract). The extraction of CAO, with six out of seven aqueous DESs, was more efficient than with 50% ethanol, resulting in CAO concentration range from 119.6 μg/mL, in Pro-sucrose 3:2 extract, to 172.4 μg/mL, in Pro-glucose 1:1 extract. Pro-fructose 1:1 extract had the lowest content of CAO (86.7 μg/mL) among the tested DESs. At the lowest tested concentration (5%), aqueous DESs did not significantly affect survival of AsPC-1 cells in comparison to the untreated cells (83.1-90.0% and 86.6% of cells remained viable, respectively). Both proline-glucose DESs demonstrated the lowest toxicity. However, at the highest concentration (25%) all aqueous DESs caused death of more than 70% of AsPC-1 cells. CONCLUSIONS: The obtained results indicate that proline and sugar/sugar alcohol based deep eutectic solvents are good extracting agents for phenolic compounds, especially for higher caffeic acid oligomers such as clinopodic acid O. Additionally, low cytotoxicity of tested DESs is a good starting predictor of their safety and potential usage.The International Bioscience Conference and the 8th International PSU – UNS Bioscience Conference - IBSC2021, Novi Sad, 25th to 26th November 202

A novel binuclear hydrazone-based Cd(II) complex is a strong pro-apoptotic inducer with significant activity against 2D and 3D pancreatic cancer stem cells

A novel binuclear Cd complex (1) with hydrazone-based ligand was prepared and characterized by spectroscopy and single crystal X-ray diffraction techniques. Complex 1 reveals a strong pro-apoptotic activity in both human, mammary adenocarcinoma cells (MCF-7) and pancreatic AsPC-1 cancer stem cells (CSCs). While apoptosis undergoes mostly caspase-independent, 1 stimulates the activation of intrinsic pathway with noteworthy down regulation of caspase-8 activity in respect to non-treated controls. Distribution of cells over mitotic division indicates that 1 caused DNA damage in both cell lines, which is confirmed in DNA interaction studies. Compared to 1, cisplatin (CDDP) does not achieve cell death in 2D cultured AsPC-1 cells, while induces different pattern of cell cycle changes and caspase activation in 2D cultured MCF-7 cells, implying that these two compounds do not share similar mechanism of action. Additionally, 1 acts as a powerful inducer of mitochondrial superoxide production with dissipated trans-membrane potential in the majority of the treated cells already after 6 h of incubation. On 3D tumors, 1 displays a superior activity against CSC model, and at 100 M induces disintegration of spheroids within 2 days of incubation. Fluorescence spectroscopy, along with molecular docking show that compound 1 binds to the minor groove of DNA. Compound 1 binds to the human serum albumin (HSA) showing that the HSA can effectively transport and store 1 in the human body. Thus, our current study strongly supports further investigations on antitumor activity of 1 as a drug candidate for the treatment of highly resistant pancreatic cancer

Evaluation of the anticarcinogenic effects of the synthetic flavagline FL3 on cancer stem cells : characterization of the molecular mechanisms involved

Il est connu aujourd'hui qu'une petite sous-population de cellules au sein des tumeurs possède une puissante capacité d'auto-renouvellement et est impliquée dans la progression tumorale, l'agressivité et la résistance à la fois à la chimiothérapie et la radiothérapie. Ces cellules, nommées cellules souches cancéreuses(CSC), sont connues pour exprimer les facteurs de souchitude Oct4 et Nanog, quand elles sont pluripotentes. Le but de ma thèse était d'analyser les effets de petites molécules pharmacologiques en mesure de cibler chez les SCCces régulateurs d'auto-renouvellement, afin d'apporter de nouvelles thérapies efficaces contre le cancer. Plus précisément, ma thèse avait pour but d'étudier l'activité anticancéreuse sélective d'une flavagline synthétique, à savoir FL3, sur un modèle de SCC peu différencié et très malin (tératocarcinome) qui exprime les facteurs de souchitude. Nous avons également utilisé un modèle de cellules souches normales restreintes (NSC) (de type fibroblastique), pour évaluer l'effet sélectif de ce médicament. Nous avons constaté que, contrairement aux NSCs, FL3 était capable de déclencher un processus pro-apoptotique mitochondriale dans les CSCs, via l'activation dep38 MAPK et de la cas pase 3, suivie par une régulation négative de Oct4 et Nanog. Nous avons ensuite étudié le mécanisme moléculaire impliqué dans la protection des NSCs contre les effets cytotoxiques de la drogue. Nous avons constaté que FL3 active sélectivement les protéines pro-survie Akt et Bad dans les NSCs. En effet, l'inhibition de la sur-expression de ces protéines a déclenché un processus pro-apoptotique lié à la caspase-3 dans les NSCs traitées par FL3. Dans une deuxième étape, nous avons montré que FL3 à faible concentration, était capable d'induire la différenciation des CSCs par la régulation négative de l'expression d'Oct4 et de Nanog, tant au niveau de la traduction que de la transcription. Cet effet a coïncidé avec une régulation à la hausse de l'expression de plusieurs marqueurs neuronaux. Pris dans leur ensemble, les résultats présentés dans ma thèse démontrent clairement que la flavagline synthétique FL3 est un composé anticancéreux puissant, agissant comme un agent sélectif pro-apoptotique et pro-différenciation sur les cellules souches cancéreuses, sans effets sur les cellules souche normales.It is believed that small subpopulation of cells within the tumor, with powerful self-renewal capacity, are involved in tumor progression, aggressiveness and resistance to both chemo- and radio-therapy. These cells, named cancer stem cells (CSCs), are known to express the stemness factors Oct4 and Nanog, when they are highly pluripotent. The aim of my thesis was therefore to analyze the effects of small pharmacological molecules which are able to target in CSCs these self-renewal regulators, in order to bring new effective therapies for cancer. More specifically, this thesis was aimed to study the selective anticancer activity of a synthetic flavagline, namely FL3,on a poorly differentiated and highly malignant CSC model (i.e. the teratocarcinomal stem-like cell) that expresses the stemness factors. Here in we also used a model of very restricted normal stem cell (NSC) (i.e. the fibroblasticstem-like cell), to evaluate the selective effect of this drug. We found that, unlike in NSCS, FL3 was able to trigger a mitochondrial pro-apoptotic process in CSCS, via the activation of p38 MAPK and caspase3, followed by a downregulation of Oct4 and Nanog. We newt investigated the molecular mechanism involved in the protection ofNSCS against the cytotoxic effects of the drug. We found that FL3 selectively activated the prosurvival proteins Akt and Bad in NSCS. Indeed, forced inhibition of the expression of these proteins triggered a caspase-3 proapoptotic process in FL3-treated NSCS. In a next step, we showed that the drug, at low concentration, was able to induce the differentiation of CSCS, by downregulating the expression of Oct4 and Nanog at both transcriptionand translation levels. This effect coincided with an upregulation of the expression of several neural markers. Taken as a whole, the results reported in my thesis clearly demonstrate that the synthetic flavagline FL3 is a powerful anticancer compound, since it acts as a selective proapoptotic and pro-differentiating agent on cancer stem-like cells, without having any effect on normal stem-like cells

Evaluation des effets anticarcinogéniques de la flavagline synthétique FL3 sur les cellules souches cancéreuses : caractérisation des mécanismes moléculaires mis en jeu

It is believed that small subpopulation of cells within the tumor, with powerful self-renewal capacity, are involved in tumor progression, aggressiveness and resistance to both chemo- and radio-therapy. These cells, named cancer stem cells (CSCs), are known to express the stemness factors Oct4 and Nanog, when they are highly pluripotent. The aim of my thesis was therefore to analyze the effects of small pharmacological molecules which are able to target in CSCs these self-renewal regulators, in order to bring new effective therapies for cancer. More specifically, this thesis was aimed to study the selective anticancer activity of a synthetic flavagline, namely FL3,on a poorly differentiated and highly malignant CSC model (i.e. the teratocarcinomal stem-like cell) that expresses the stemness factors. Here in we also used a model of very restricted normal stem cell (NSC) (i.e. the fibroblasticstem-like cell), to evaluate the selective effect of this drug. We found that, unlike in NSCS, FL3 was able to trigger a mitochondrial pro-apoptotic process in CSCS, via the activation of p38 MAPK and caspase3, followed by a downregulation of Oct4 and Nanog. We newt investigated the molecular mechanism involved in the protection ofNSCS against the cytotoxic effects of the drug. We found that FL3 selectively activated the prosurvival proteins Akt and Bad in NSCS. Indeed, forced inhibition of the expression of these proteins triggered a caspase-3 proapoptotic process in FL3-treated NSCS. In a next step, we showed that the drug, at low concentration, was able to induce the differentiation of CSCS, by downregulating the expression of Oct4 and Nanog at both transcriptionand translation levels. This effect coincided with an upregulation of the expression of several neural markers. Taken as a whole, the results reported in my thesis clearly demonstrate that the synthetic flavagline FL3 is a powerful anticancer compound, since it acts as a selective proapoptotic and pro-differentiating agent on cancer stem-like cells, without having any effect on normal stem-like cells.Il est connu aujourd'hui qu'une petite sous-population de cellules au sein des tumeurs possède une puissante capacité d'auto-renouvellement et est impliquée dans la progression tumorale, l'agressivité et la résistance à la fois à la chimiothérapie et la radiothérapie. Ces cellules, nommées cellules souches cancéreuses(CSC), sont connues pour exprimer les facteurs de souchitude Oct4 et Nanog, quand elles sont pluripotentes. Le but de ma thèse était d'analyser les effets de petites molécules pharmacologiques en mesure de cibler chez les SCCces régulateurs d'auto-renouvellement, afin d'apporter de nouvelles thérapies efficaces contre le cancer. Plus précisément, ma thèse avait pour but d'étudier l'activité anticancéreuse sélective d'une flavagline synthétique, à savoir FL3, sur un modèle de SCC peu différencié et très malin (tératocarcinome) qui exprime les facteurs de souchitude. Nous avons également utilisé un modèle de cellules souches normales restreintes (NSC) (de type fibroblastique), pour évaluer l'effet sélectif de ce médicament. Nous avons constaté que, contrairement aux NSCs, FL3 était capable de déclencher un processus pro-apoptotique mitochondriale dans les CSCs, via l'activation dep38 MAPK et de la cas pase 3, suivie par une régulation négative de Oct4 et Nanog. Nous avons ensuite étudié le mécanisme moléculaire impliqué dans la protection des NSCs contre les effets cytotoxiques de la drogue. Nous avons constaté que FL3 active sélectivement les protéines pro-survie Akt et Bad dans les NSCs. En effet, l'inhibition de la sur-expression de ces protéines a déclenché un processus pro-apoptotique lié à la caspase-3 dans les NSCs traitées par FL3. Dans une deuxième étape, nous avons montré que FL3 à faible concentration, était capable d'induire la différenciation des CSCs par la régulation négative de l'expression d'Oct4 et de Nanog, tant au niveau de la traduction que de la transcription. Cet effet a coïncidé avec une régulation à la hausse de l'expression de plusieurs marqueurs neuronaux. Pris dans leur ensemble, les résultats présentés dans ma thèse démontrent clairement que la flavagline synthétique FL3 est un composé anticancéreux puissant, agissant comme un agent sélectif pro-apoptotique et pro-différenciation sur les cellules souches cancéreuses, sans effets sur les cellules souche normales

Three dimensional in vitro culture systems in anticancer drug discovery targeted on cancer stem cells

International audienceWorldwide, tumors are one of the most common causes of death. Every year 3.7 million new cases occur in Europe and more than 1.9 million patients die (WHO data). Most of the fields of research are focused on developing new therapeutic strategies that will be effective in eliminating the tumor, preventing its remission, and avoiding or reducing the side effects of therapy. In the past, generally classical 2D cell cultures or immunodeficient animal models had been used to cultivate and test drugs on human cancer cell lines. Nowadays, there are increasing interests in three-dimensional (3D) cell cultures, a method with significant differences from flat cultured cells, both considering gene expressions and cell-cell interactions. Various evidence suggests that high tumorigenic properties might be dependent on the occurrence of a small cell population, pointed out to be responsible for metastasis and recurrence. This population is called cancer stem cells (CSCs), hinted to have a lot of similarities with normal stem cells. CSCs are the main reason for chemotherapy failure as well as multi-drug resistance (MDR). CSCs can also interact through the cytokine network, with other cells like the macrophages of the inflammatory system. The big advantage of a 3D culture is the possibility to isolate and investigate the CSCs population surrounded by its environment. This article aims to sum up known 3D cell cultures, especially in the field of CSCs research due to the importance of the tumor's environment on stem cell's markers expression and their development

The synthetic flavagline FL3 spares normal human skin cells from its cytotoxic effect via an activation of Bad

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Biological Activities of Aqueous Extracts from Carob Plant (Ceratonia siliqua L.) by Antioxidant, Analgesic and Proapoptotic Properties Evaluation

International audienceThe present work describes the volatile compounds profile and phytochemical content of Ceratonia siliqua L. Fifty different components have been identified. Among them, three constituents are shared i.e., 2-methlybutanoic acid, methyl hexanoate and limonene by different common carob preparations: pulp decoction (PD), seeds decoction (SD) and Rob, a sweet syrup extracted from the pulp of the carob pod. Each extract exhibits different volatile aromatic emission profiles. The antioxidant activity of the extracts was evaluated using three methods, DPPH, ABTS and FRAP, producing a dose-dependent response. The IC 50, when determined by FRAP, gave the lowest values (0.66 ± 0.01, 0.73 ± 0.05 and 0.55 ± 0.00 mg/mL PD, SD and Rob, respectively). The nociception essay, after intraperitoneal injection of acetic acid in mice, demonstrated that Rob, pulp and seeds decoction extracts showed an efficient inhibition of writhes over time, with persistence over 30 min. The SD decoction revealed the highest efficacy in decreasing the writhing reflex (90.3 ± 1.2%; p < 0.001). Furthermore, the proapoptotic activity of SD against three human cell line, THP-1, MCF-7 and LOVO, evaluated by flow cytometry, showed a significantly stronger proapoptotic activity on colon cancer (LOVO) than on the other cell lines, a phenomenon known as phenotypic selectivity

Cytotoxic and Pro-Apoptotic Effects of Leaves Extract of <i>Antiaris africana</i> Engler (<i>Moraceae</i>)

Antiaris africana Engler leaves have been used in Senegalese folk medicine to treat breast cancer. The present study aimed to investigate the anticancer potential of Antiaris africana Engler leaves using several human cancer cell lines. The leaves of Antiaris africana Engler were extracted in parallel with water or 70% ethanol and each extract divided into three parts by successive liquid–liquid extraction with ethyl acetate and butanol. The phytochemical components of the active extract were investigated using ultra-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (UPLC-DAD-QTOF-MS/MS). The cytotoxic and cytostatic effects of each extract, as well as their fractions, were evaluated in vitro via flow and image cytometry on different human cancer phenotypes, such as breast (MCF-7), pancreas (AsPC-1), colon (SW-620) and acute monocytic leukemia (THP-1). Both hydro-alcoholic and aqueous extracts induced strong apoptosis in MCF-7 cells. The water fraction of the hydro-alcoholic extract was found to be the most active, suppressing the cell growth of MCF-7 in a dose-dependent manner. The half maximum effective concentration (EC50) of this fraction was 64.6 ± 13.7 μg/mL for MCF-7, with equivalent values for all tested phenotypes. In parallel, the apoptotic induction by this fraction resulted in a EC50 of 63.5 ± 1.8 μg/mL for MCF-7, with again equivalent values for all other cellular tested phenotypes. Analysis of this fraction by UPLC-DAD-QTOF-MS/MS led to the identification of hydroxycinnamates as major components, one rutin isomer, and three cardiac glycosides previously isolated from seeds and bark of Antiaris africana Engler and described as cytotoxic in human cancer models. These results provide supportive data for the use of Antiaris africana Engler leaves in Senegal

Anti-Hyperalgesic Properties of Menthol and Pulegone

International audienceContext: Menthol, the main monoterpene found in Mentha piperita L. (M. piperita) is known to modulate nociceptive threshold and is present in different curative preparations that reduce sensory hypersensitivities in pain conditions. While for pulegone, a menthol-like monoterpene, only a limited number of studies focus on its putative analgesic effects, pulegone is the most abundant monoterpene present in Calamintha nepeta (L.) Savi (C. nepeta), a plant of the Lamiaceae family used in traditional medicine to alleviate rheumatic disorders, which counts amongst chronic inflammatory diseases.Objectives: Here, we analyzed the monoterpenes composition of C. nepeta and M. piperita. We then compared the putative anti-hyperalgesic effects of the main monoterpenes found, menthol and pulegone, in acute inflammatory pain conditions.Methods: C. nepeta and M. piperita extracts were obtained through pressurized liquid extraction and analyzed by gas chromatography-mass spectrometry. The in vitro antiinflammatory activity of menthol or pulegone was evaluated by measuring the secretion of the tumour necrosis factor alpha (TNF α) from LPS-stimulated THP-1 cells. The in vivo antihyperalgesic effects of menthol and pulegone were tested on a rat inflammatory pain model.Results: Pulegone and menthol are the most abundant monoterpene found in C. nepeta (49.41%) and M. piperita (42.85%) extracts, respectively. In vitro, both pulegone and menthol act as strong anti-inflammatory molecules, with EC50 values of 1.2 ± 0.2 and 1.5 ± 0.1 mM, respectively, and exert cytotoxicity with EC50 values of 6.6 ± 0.3 and 3.5 ± 0.2 mM, respectively