A Preliminary Report Regarding the Morphological Changes of Nano-Enabled Pharmaceutical Formulation on Human Lung Carcinoma Monolayer and 3D Bronchial Microtissue.

Background and Objectives: Nowadays, the development of enabled pharmaceutical nanoparticles of solid lipid type is continuously growing, because they have the potential to be used for targeted drug release leading to an increased effect of chemotherapy, being used in lung cancer nano-diagnosis and nano-therapy. The current study reports the preliminary results obtained regarding the biological effect of a new nano-enabled pharmaceutical formulation in terms of its cytotoxic and biosafety profile. Materials and Methods: The pharmaceutical formulations consist of solid lipid nanoparticles (SLN) obtained via the emulsification-diffusion method by loading green iron oxide nanoparticles (green-IONPs) with a pentacyclic triterpene (oleanolic acid-OA). Further, a complex biological assessment was performed, employing three-dimensional (3D) bronchial microtissues (EpiAirwayTM) to determine the biosafety profile of the SLN samples. The cytotoxic potential of the samples was evaluated on human lung carcinoma, using an in vitro model (A549 human lung carcinoma monolayer). Results: The data revealed that the A549 cell line was strongly affected after treatment with SLN samples, especially those that contained OA-loaded green-IONPs obtained with Ocimum basilicum extract (under 30% viability rates). The biosafety profile investigation of the 3D normal in vitro bronchial model showed that all the SLN samples negatively affected the viability of the bronchial microtissues (below 50%). As regards the morphological changes, all the samples induce major changes such as loss of the surface epithelium integrity, loss of epithelial junctions, loss of cilia, hyperkeratosis, and cell death caused by apoptosis. Conclusions: In summary, the culprit for the negative impact on viability and morphology of 3D normal bronchial microtissues could be the too-high dose (500 µg/mL) of the SLN sample used. Nevertheless, further adjustments in the SLN synthesis process and another complex in vitro evaluation will be considered for future research.

Phytochemical and Nutraceutical Screening of Ethanol and Ethyl Acetate Phases of Romanian Galium verum Herba (Rubiaceae).

Galium species are used worldwide for their antioxidant, antibacterial, antifungal, and antiparasitic properties. Although this plant has demonstrated its antitumor properties on various types of cancer, its biological activity on cutaneous melanoma has not been established so far. Therefore, the present study was designed to investigate the phytochemical profile of two extracts of G. verum L. herba (ethanolic and ethyl acetate) as well as the biological profile (antioxidant, antimicrobial, and antitumor effects) on human skin cancer. The extracts showed similar FT-IR phenolic profiles (high chlorogenic acid, isoquercitrin, quercitrin, and rutin), with high antioxidant capacity (EC50 of ethyl acetate phase (0.074 ± 0.01 mg/mL) > ethanol phase (0.136 ± 0.03 mg/mL)). Both extracts showed antimicrobial activity, especially against Gram-positive Streptococcus pyogenes and Staphylococcus aureus bacilli strains, the ethyl acetate phase being more active. Regarding the in vitro antitumor test, the results revealed a dose-dependent cytotoxic effect against A375 melanoma cell lines, more pronounced in the case of the ethyl acetate phase. In addition, the ethyl acetate phase stimulated the proliferation of human keratinocytes (HaCaT), while this effect was not evident in the case of the ethanolic phase at 24 h post-stimulation. Consequently, G. verum l. could be considered a promising phytocompound for the antitumor approach of cutaneous melanoma.

Antimicrobial activity, in vitro anticancer effect (MCF-7 breast cancer cell line), antiangiogenic and immunomodulatory potentials of Populus nigra L. buds extract.

PURPOSE: The aim of this study was to evaluate the antioxidant potential, antimicrobial activity, the in vitro anticancer effect (tested on MCF-7 breast cancer cell line), as well as the antiangiogenic and immunomodulatory potential of Populus nigra L. bud (Pg) extract collected from the western part of Romania. RESULTS: Populus nigra L. bud extract presents an important antioxidant activity, due to the rich phytochemical composition. Regarding the biological activity, results have shown that poplar bud extract presents a significant inhibitory activity against Gram-positive bacteria and a dose-dependent decrease of MCF-7 tumor cell viability with an IC50 of 66.26 µg/mL, while not affecting healthy cells. Phenomena of early apoptotic events at the maximum concentration tested (150 µg/mL) were detected by Annexin V-PI double staining. The extract induced G0/G1 phase cell cycle arrest. In addition, Pg extract showed antiangiogenic potential on the chorioallantoic membrane. Also, at the highest concentration (150 µg/mL), good tolerability and no signs of toxicity upon vascular plexus were observed. Moreover, in low concentrations, the Pg extract had immunomodulatory activity on primary human dendritic cells by upregulating IL-12 and IL-23 subunits. CONCLUSION: The study concludes that poplar bud extract elicited antioxidant activity, antitumor properties on the breast cancer cell line, followed by an antiangiogenic effect and an immunomodulatory potential on human primary dendritic cells. The biological activity of Populus nigra L. buds extract may open new directions of research on the topic addressed.

Melissa officinalis L. Aqueous Extract Exerts Antioxidant and Antiangiogenic Effects and Improves Physiological Skin Parameters.

Melissa officinalis (MO) is a medicinal plant well-known for its multiple pharmacological effects, including anti-inflammatory, anticancer and beneficial effects on skin recovery. In this context, the present study was aimed to investigate the in vitro and in vivo safety profile of an MO aqueous extract by assessing cell viability on normal (HaCaT-human keratinocytes) and tumor (A375-human melanoma) cells and its impact on physiological skin parameters by a non-invasive method. In addition, the antioxidant activity and the antiangiogenic potential of the extract were verified. A selective cytotoxic effect was noted in A375 cells, while no toxicity was noticed in healthy cells. The MO aqueous extract safety profile after topical application was investigated on SKH-1 mice, and an enhanced skin hydration and decreased erythema and transepidermal water loss levels were observed. The in ovo CAM assay, performed to investigate the potential modulating effect on the angiogenesis process and the blood vessels impact, indicated that at concentrations of 100 and 500 µg/mL, MO aqueous extract induced a reduction of thin capillaries. No signs of vascular toxicity were recorded at concentrations as high as 1000 µg/mL. The aqueous extract of MO leaves can be considered a promising candidate for skin disorders with impaired physiological skin parameters.

Correlations on Phenolic Screening Related to In Vitro and In Ovo Assessment of Ocimum basilicum L. Hydro-Alcoholic Extracts Used as Skin Active Ingredient.

The current study was aimed to evaluate the phenolic composition parameters of two hydro-alcoholic extracts of Ocimum basilicum L. (OB) obtained from the aerial part (without leaves) and leaves, in order to determine their contribution to the antioxidant activity (AOA). Both hydro-alcoholic extracts have proven to be rich in polyphenolic compounds, flavonoids, flavonols and tannins. Therefore, the leaves' extracts reveal an inhibition percentage of 89%, almost comparable with the standard reference (95%). To complete the toxicological profile, the study also assessed the potential cytotoxicity of basil hydro-alcoholic extracts on immortalized human keratinocytes (HaCaT), skin human fibroblasts (1BR3), mice epidermis (JB6Cl41-5a) and primary human melanocytes (HEMa) cells, correlated to A375 antitumor in vitro activity. The extracts did not induce significant cytotoxic effect on any of the selected normal cell lines but showed relevant activity on A375 cells. Considering the low values obtained regarding the irritative effects in the chorionallantoic membrane of the egg on blood vessels, we can emphasize that both extracts can be considered as biocompatible ingredients. Regarding the potential activity of hydro-alcoholic extracts on human skin, the decrease of erythema values after the application of extracts was a relevant observation which indicates the anti-inflammatory potential of Ocimum basilicum L.

Thermosensitive Betulinic Acid-Loaded Magnetoliposomes: A Promising Antitumor Potential for Highly Aggressive Human Breast Adenocarcinoma Cells Under Hyperthermic Conditions.

PURPOSE: Breast cancer presents one of the highest rates of prevalence around the world. Despite this, the current breast cancer therapy is characterized by significant side effects and high risk of recurrence. The present work aimed to develop a new therapeutic strategy that may improve the current breast cancer therapy by developing a heat-sensitive liposomal nano-platform suitable to incorporate both anti-tumor betulinic acid (BA) compound and magnetic iron nanoparticles (MIONPs), in order to address both remote drug release and hyperthermia-inducing features. To address the above-mentioned biomedical purposes, the nanocarrier must possess specific features such as specific phase transition temperature, diameter below 200 nm, superparamagnetic properties and heating capacity. Moreover, the anti-tumor activity of the developed nanocarrier should significantly affect human breast adenocarcinoma cells. METHODS: BA-loaded magnetoliposomes and corresponding controls (BA-free liposomes and liposomes containing no magnetic payload) were obtained through the thin-layer hydration method. The quality and stability of the multifunctional platforms were physico-chemically analysed by the means of RAMAN, scanning electron microscopy-EDAX, dynamic light scattering, zeta potential and DSC analysis. Besides this, the magnetic characterization of magnetoliposomes was performed in terms of superparamagnetic behaviour and heating capacity. The biological profile of the platforms and controls was screened through multiple in vitro methods, such as MTT, LDH and scratch assays, together with immunofluorescence staining. In addition, CAM assay was performed in order to assess a possible anti-angiogenic activity induced by the test samples. RESULTS: The physico-chemical analysis revealed that BA-loaded magnetoliposomes present suitable characteristics for the purpose of this study, showing biocompatible phase transition temperature, a diameter of 198 nm, superparamagnetic features and heating capacity. In vitro results showed that hyperthermia induces enhanced anti-tumor activity when breast adenocarcinoma MDA-MB-231 cells were exposed to BA-loaded magnetoliposomes, while a low cytotoxic rate was exhibited by the non-tumorigenic breast epithelial MCF 10A cells. Moreover, the in ovo angiogenesis assay endorsed the efficacy of this multifunctional platform as a good strategy for breast cancer therapy, under hyperthermal conditions. Regarding the possible mechanism of action of this multifunctional nano-platform, the immunocytochemistry of the MCF7 and MDA-MB-231 breast carcinoma cells revealed a microtubule assembly modulatory activity, under hyperthermal conditions. CONCLUSION: Collectively, these findings indicate that BA-loaded magnetoliposomes, under hyperthermal conditions, might serve as a promising strategy for breast adenocarcinoma treatment.

Identification of Resveratrol as Bioactive Compound of Propolis from Western Romania and Characterization of Phenolic Profile and Antioxidant Activity of Ethanolic Extracts.

The present study aimed to assess the phenolic content of eight ethanolic propolis samples (P1-P8) harvested from different regions of Western Romania and their antioxidant activity. The mean value of total phenolic content was 214 ± 48 mg gallic acid equivalents (GAE)/g propolis. All extracts contained kaempferol (514.02 ± 114.80 µg/mL), quercetin (124.64 ± 95.86 µg/mL), rosmarinic acid (58.03 ± 20.08 µg/mL), and resveratrol (48.59 ± 59.52 µg/mL) assessed by LC-MS. The antioxidant activity was evaluated using 2 methods: (i) DPPH (2,2-diphenyl-1-picrylhydrazyl) assay using ascorbic acid as standard antioxidant and (ii) FOX (Ferrous iron xylenol orange OXidation) assay using catalase as hydrogen peroxide (H2O2) scavenger. The DPPH radical scavenging activity was determined for all samples applied in 6 concentrations (10, 5, 3, 1.5, 0.5 and 0.3 mg/mL). IC50 varied from 0.0700 to 0.9320 mg/mL (IC50 of ascorbic acid = 0.0757 mg/mL). The % of H2O2 inhibition in FOX assay was assessed for P1, P2, P3, P4 and P8 applied in 2 concentrations (5 and 0.5 mg/mL). A significant H2O2% inhibition was obtained for these samples for the lowest concentration. We firstly report the presence of resveratrol as bioactive compound in Western Romanian propolis. The principal component analysis revealed clustering of the propolis samples according to the polyphenolic profile similarity.

Romanian Wormwood (Artemisia absinthium L.): Physicochemical and Nutraceutical Screening.

Artemisia species are used worldwide for their antioxidant, antimicrobial and anti-inflammatory properties. This research was designed to investigate the phytochemical profile of two ethanolic extracts obtained from leaves and stems of A. absinthium L. as well as the biological potential (antioxidant activity, cytotoxic, anti-migratory and anti-inflammatory properties). Both plant materials showed quite similar thermogravimetric, FT-IR phenolic profile (high chlorogenic acid) with mild antioxidant capacity [ascorbic acid (0.02-0.1) > leaves (0.1-2.0) > stem (0.1-2.0)]. Alcoholic extracts from these plant materials showed a cytotoxic effect against A375 (melanoma) and MCF7 (breast adenocarcinoma) and affected less the non-malignant HaCaT cells (human keratinocytes) at 72 h post-stimulation and this same trend was observed in the anti-migratory (A375, MCF7 > HaCat) assay. Lastly, extracts ameliorated the pro-inflammatory effect of TPA (12-o-tetradecanoylphorbol-13-acetate) in mice ears, characterized by a diffuse neutrophil distribution with no exocytosis or micro-abscesses.

A Comparative Study of Melissa officinalis Leaves and Stems Ethanolic Extracts in terms of Antioxidant, Cytotoxic, and Antiproliferative Potential.

Melissa officinalis L. has attracted an increased interest in recent years due to its multiple pharmacological effects. This study aimed to compare two M. officinalis ethanolic extracts, obtained from leaves and stems, with regard to their antioxidant activity, total phenolic content, and cytotoxic effects. M. officinalis ethanolic extracts showed a very good antioxidant activity in the DPPH test, correlated with the content in total phenols: higher in the case of M. officinalis from leaves extract (32.76 mg GAE/g) and lower for M. officinalis from stems extract (8.4 mg GAE/g). The lemon balm extracts exerted a cytotoxic effect on breast cancer cells (MDA-MB-231) even at low concentrations (100 µg/mL), whereas, in the case of healthy HaCat cells, M. officinalis leaves extract only displayed cytotoxicity at much higher concentrations (500 and 1000 µg/mL) and M. officinalis stems extracts were highly cytotoxic (starting at 100 µg/mL). In addition, the extracts exerted inhibitory effects on cell migration and proliferation. These results provide information that confirms the high potential of M. officinalis as a source of chemopreventive agents. Moreover, these data can be considered a solid background for further in vivo studies involving mice bearing breast tumors.

Main Isoflavones Found in Dietary Sources as Natural Anti-inflammatory Agents.

BACKGROUND: Isoflavones are natural compounds belonging to the class of isoflavonoids, molecules with a chemical structure based on the 3-phenyl chromen-4-one backbone. As a particular element, the isoflavones are also called phytoestrogens, due to the structural similarity with 17-ß estradiol, the primary female sex hormone, presenting both estrogen-agonist and estrogen-antagonist properties. This class of secondary plant metabolites has been extensively reported in the literature for a wide range of therapeutic activities including antioxidant, chemopreventive, anti-inflammatory, antiallergic, antibacterial, and cardio preventive effects. OBJECTIVE: To sum up the latest information regarding the main isoflavones found in dietary sources as natural anti-inflammatory agents. RESULTS: The review updates with the recent research about genistein, daidzein, glycitein, biochanin A, formononetin and equol as anti-inflammatory phytocompounds. As a particular element, the implications of these isoflavones in the link between inflammation and angiogenesis are also discussed. CONCLUSION: The review concludes that the main isoflavones in dietary sources display in vitro and/or in vivo anti inflammatory potential by activating various biochemical and molecular mechanisms. The depicted findings translated into practical aspects strengthen the idea that the isoflavones genistein, daidzein, glycitein, biochanin A, formononetin and equol can be used in various inflammation based diseases as an alternative source for classical medication and that consummation of foods rich in isoflavones may represent prerequisite use in order to prevent the evolution of inflammation based diseases.