Insight into the influence of natural deep eutectic solvents on the extraction of phenolic compounds from poplar type propolis: Composition and in vitro biological activity.

Natural deep eutectic solvents (NADESs) have been considered promising to replace traditional volatile and toxic organic solvents for the extraction of biologically active substances from natural sources. This work applied an efficient and ethanol-exclusion strategy for extraction of phenolic compounds from poplar type propolis using five known NADESs (lactic acid:1,2-propanediol 1:1, lactic acid:fructose 5:1, choline chloride:1,2-propanediol 1:3, choline chloride:1,2-propanediol:water 1:1:1 and betaine:malic acid:water 1:1:6). The selected NADESs' extractability was evaluated by measuring the concentrations of total phenolics and total flavones and flavonols in the propolis extracts obtained, which qualitative chemical composition was further determined in detail by gas chromatography-mass spectrometry (GC-MS) analysis. It demonstrated that the chemical profiles of NADES and 70% ethanolic propolis extracts are similar. To expand the knowledge about the role of the applied solvents in the poplar propolis extraction process, the in vitro antimicrobial, cytotoxic and genotoxic activity of both NADESs and liquid NADES extracts were evaluated. The results revealed that the use of the selected NADESs as an extraction media for phenolic compounds from poplar propolis not only delivered a good extraction yield in some cases, but generally led to the preservation of propolis extracts' biological activity and even to the enhancement of their antimicrobial effect in comparison with the hydroethanolic one. Besides, the tested NADESs except for lactic acid:fructose and betaine:malic acid:water exerted low to negligible toxicity against normal cells treated and apart from lactic acid:fructose the remaining solvents demonstrated concentration-dependent moderate to subtle genotoxicity. There is a probability that not the supramolecular structure of the NADESs, but their components, played a key role for the observed biological effects. The present study has demonstrated an alternative approach for extracting the biologically active complex from poplar type propolis using NADESs, which could be useful for further pharmaceutical and cosmeceutical applications.

Functional Hydrogels for Delivery of the Proteolytic Enzyme Serratiopeptidase.

Hydrogels are superior wound dressings because they can provide protection and hydration of the wound, as well as the controlled release of therapeutic substances to aid tissue regeneration and the healing process. Hydrogels obtained from natural precursors are preferred because of their low cost, biocompatibility, and biodegradability. We describe the synthesis of novel functional hydrogels based on two natural products-citric acid (CA) and pentane-1,2,5-triol (PT, a product from lignocellulose processing) and poly(ethylene glycol) (PEG-600)-via an environment friendly approach. The hydrogels were prepared via monomer crosslinking through a polycondensation reaction at an elevated temperature in the absence of any solvent. The reagents were blended at three different compositions with molar ratios of hydroxyl (from PT and PEG) to carboxyl (from CA) groups of 1:1, 1:1.4, and 1.4:1, respectively. The effect of the composition on the physicomechanical properties of materials was investigated. All hydrogels exhibited pH-sensitive behavior, while the swelling degree and elastic modulus were dependent on the composition of the polymer network. The proteolytic enzyme serratiopeptidase (SER) was loaded into a hydrogel via physical absorption as a model drug. The release profile of SER and the effects of the enzyme on healthy skin cells were assessed. The results showed that the hydrogel carrier could provide the complete release of the loaded enzyme.

Polycarbonate-Based Copolymer Micelles as Biodegradable Carriers of Anticancer Podophyllotoxin or Juniper Extracts.

Podophyllotoxin (PPT) is used in the industrial production of efficient anticancer, antiviral and other drugs. Sinopodophyllum hexandrum or Podophyllum peltatum are natural sources of PPT, but at present they are considered as endangered species. Their PPT content is variable, depending on the growing conditions. Searching for new sources of PPT, some representatives of the genus Juniperus were found to exhibit efficient PPT biosynthesis. However, PPT is highly toxic and poorly soluble in water compound, which limits its clinical applications. In this connection, amphiphilic polymer micelles are considered to be suitable PPT carriers, aimed at increase in water solubility and decrease in toxicity. The present research deals with the evaluation of MPEG-polycarbonate block copolymer micelles loaded with PPT or juniper extracts. The active component-loaded polymer nanocarriers were characterized by dynamic and electrophoretic light scattering, as well as by transmission electron microscopy. The active component loading efficiency and loading capacity were also determined. Highly efficient antiproliferative activity of the loaded micelles was determined in a panel of cancer cell lines. The obtained amphiphilic nanocarriers, loaded with PPT-containing bioactive components, have application in future in vivo preclinical trials of their pharmacokinetics and pharmacodynamics as potential therapeutical agents in the prospective nanomedicine.

Biopolymeric Nanogel as a Drug Delivery System for Doxorubicin-Improved Drug Stability and Enhanced Antineoplastic Activity in Skin Cancer Cells.

In this study, doxorubicin was loaded in a chitosan-albumin nanogel with the aim of improving its stability and exploring the potential of the system in the treatment of skin cancer. Infrared spectroscopy and X-ray diffraction confirmed the encapsulation of the drug. Transmission electron microscopy revealed the spherical shape of the nanogel particles. The drug-loaded nanogel was characterized with a small diameter of 29 nm, narrow polydispersity (0.223) and positive zeta potential (+34 mV). The exposure of encapsulated doxorubicin to light (including UV irradiation and daylight) did not provoke any degradation, whereas the nonencapsulated drug was significantly degraded. In vitro studies on keratinocytes (HaCaT) and epidermoid squamous skin carcinoma cells (A-431) disclosed that the encapsulated doxorubicin was more cytotoxic on both cell lines than the pure drug was. More importantly, the cytotoxic concentration of encapsulated doxorubicin in carcinoma cells was approximately two times lower than that in keratinocytes, indicating that it would not affect them. Thus, the loading of doxorubicin into the developed chitosan-albumin nanogel definitely stabilized the drug against photodegradation and increased its antineoplastic effect on the skin cancer cell line.

Micellar Curcumin Substantially Increases the Antineoplastic Activity of the Alkylphosphocholine Erufosine against TWIST1 Positive Cutaneous T Cell Lymphoma Cell Lines.

Cutaneous T-cell lymphoma (CTCL) is a rare form of cancer with local as well as systemic manifestations. Concomitant bacterial infections increase morbidity and mortality rates due to impaired skin barrier and immune deficiency. In the current study, we demonstrated that the in vitro anti-lymphoma potential of erufosine is diminished by TWIST1 expression and micellar curcumin substantially increases its antineoplastic activity. Pharmacokinetic analysis showed that the micellar curcumin (MCRM) used in our study was characterized by low zeta potential, slow release of curcumin, and fast cell membrane penetration. The combination ratio 1:4 [erufosine:MCRM] achieved strong synergism by inhibiting cell proliferation and clonogenicity. The combined antiproliferative effects were calculated using the symbolic mathematical software MAPLE 15. The synergistic combination strongly decreased the expression of TWIST1 and protein kinase B/Akt as proven by western blotting. Significant reductions in NF-κB activation, induction of apoptosis, and altered glutathione levels were demonstrated by corresponding assays. In addition, the synergistic combination enhanced the anti-staphylococcal activity and prevented biofilm formation, as shown by crystal violet staining. Taken together, the above results show that the development of nanotechnological treatment modalities for CTCL, based on rational drug combinations exhibiting parallel antineoplastic and antibacterial effects, may prove efficacious.

Improvement of the Antimicrobial Activity of Oregano Oil by Encapsulation in Chitosan-Alginate Nanoparticles.

Oregano oil (OrO) possesses well-pronounced antimicrobial properties but its application is limited due to low water solubility and possible instability. The aim of this study was to evaluate the possibility to incorporate OrO in an aqueous dispersion of chitosan-alginate nanoparticles and how this will affect its antimicrobial activity. The encapsulation of OrO was performed by emulsification and consequent electrostatic gelation of both polysaccharides. OrO-loaded nanoparticles (OrO-NP) have small size (320 nm) and negative charge (-25 mV). The data from FTIR spectroscopy and XRD analyses reveal successful encapsulation of the oil into the nanoparticles. The results of thermogravimetry suggest improved thermal stability of the encapsulated oil. The minimal inhibitory concentrations of OrO-NP determined on a panel of Gram-positive and Gram-negative pathogens (ISO 20776-1:2006) are 4-32-fold lower than those of OrO. OrO-NP inhibit the respiratory activity of the bacteria (MTT assay) to a lower extent than OrO; however, the minimal bactericidal concentrations still remain significantly lower. OrO-NP exhibit significantly lower in vitro cytotoxicity than pure OrO on the HaCaT cell line as determined by ISO 10993-5:2009. The irritation test (ISO 10993-10) shows no signs of irritation or edema on the application site. In conclusion, the nanodelivery system of oregano oil possesses strong antimicrobial activity and is promising for development of food additives.

Prevalence of Antibiotic-Resistant Escherichia coli Isolated from Swine Faeces and Lagoons in Bulgaria.

Antimicrobial resistance (AMR) is a worldwide health problem affecting humans, animals, and the environment within the framework of the "One Health" concept. The aim of our study was to evaluate the prevalence of pathogenic strains of the species Escherichia coli (E. coli), their AMR profile, and biofilm-forming potential. The isolated strains from three swine faeces and free lagoons (ISO 16654:2001/Amd 1:2017) were confirmed using Phoenix M50 and 16S rDNA PCR. The antibiotic sensitivity to 34 clinically applied antibiotics was determined by Phoenix M50 and the disc diffusion method, according to the protocols of the CLSI and EUCAST. We confirmed the presence of 16 E. coli isolates, of which 87.5% were multi-drug-resistant and 31.25% performed strong biofilms. The possibility for the carrying and transmission of antibiotic-resistance genes to quinolones (qnr), aminoglycosides (aac(3)), ß-lactamase-producing plasmid genes ampC, and blaSHV/blaTEM was investigated. We confirmed the carrying of blaSHV/blaTEM in one and ampC in seven isolates. The strains were negative for the virulence genes (ETEC (LT, STa, and F4), EPEC (eae), and STEC/VTEC (stx and stx2all)). The results should contribute to the development of effective measures for limitation and control on the use of antibiotics, which is a key point in the WHO action plan.

In Vitro Antineoplastic and Antiviral Activity and In Vivo Toxicity of Geum urbanum L. Extracts.

This study evaluated the in vitro antineoplastic and antiviral potential and in vivo toxicity of twelve extracts with different polarity obtained from the herbaceous perennial plant Geum urbanum L. (Rosaceae). In vitro cytotoxicity was determined by ISO 10993-5/2009 on bladder cancer, (T-24 and BC-3C), liver carcinoma (HEP-G2) and normal embryonic kidney (HEK-293) cell lines. The antineoplastic activity was elucidated through assays of cell clonogenicity, apoptosis induction, nuclear factor kappa B p65 (NFκB p65) activation and total glutathione levels. Neutral red uptake study was applied for antiviral activity. The most promising G. urbanum extract was analyzed by UHPLC-HRMS. The acute in vivo toxicity analysis was carried out following OEDC 423. The ethyl acetate extract of aerial parts (EtOAc-AP) exhibited the strongest antineoplastic activity on bladder cancer cell lines (IC50 = 21.33-25.28 µg/mL) by inducing apoptosis and inhibiting NFκB p65 and cell clonogenicity. EtOAc and n-butanol extracts showed moderate antiviral activity against human adenovirus type 5 and human simplex virus type I. Seventy four secondary metabolites (gallic and ellagic acid derivatives, phenolic acids, flavonoids, etc.) were identified in EtOAc-AP by UHPLC-HRMS. This extract induced no signs of acute toxicity in liver and kidney specimens of H-albino mice in doses up to 210 mg/kg. In conclusion, our study contributes substantially to the detailed pharmacological characterization of G. urbanum, thus helping the development of health-promoting phytopreparations.

Extracts of medicinal plants with natural deep eutectic solvents: enhanced antimicrobial activity and low genotoxicity.

Natural deep eutectic solvents (NADES) are a new alternative to toxic organic solvents. Their constituents are primary metabolites, non-toxic, biocompatible and sustainable. In this study four selected NADES were applied for the extraction of two medicinal plants: Sideritis scardica, and Plantago major as an alternative to water-alcohol mixtures, and the antimicrobial and genotoxic potential of the extracts were studied. The extraction efficiency was evaluated by measuring the extracted total phenolics, and total flavonoids. Best extraction results for total phenolics for the studied plants were obtained with choline chloride-glucose 5:2 plus 30% water; but surprisingly these extracts were inactive against all tested microorganisms. Extracts with citric acid-1,2-propanediol 1:4 and choline chloride-glycerol 1:2 showed good activity against S. pyogenes, E. coli, S. aureus, and C. albicans. Low genotoxicity and cytotoxicity were observed for all four NADES and the extracts with antimicrobial activity. Our results confirm the potential of NADESs for extraction of bioactive constituents of medicinal plants and further suggest that NADES can improve the effects of bioactive extracts. Further studies are needed to clarify the influence of the studied NADES on the bioactivity of dissolved substances, and the possibility to use such extracts in the pharmaceutical and food industry.

Antineoplastic effect of a novel nanosized curcumin on cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) are a group of heterogeneous, life-threatening, extra-nodal and lymphoproliferative T cell neoplasms. Since chronic inflammation serves a key role in CTCL progression, curcumin, a natural pigment with proven anti-inflammatory and antineoplastic properties, as well as minimal toxicity, may be used as a therapeutic agent. In the present study, two formulations of curcumin (standard ethanolic and a Pluronic®P-123/F-127 micellar solution) were compared regarding their cytotoxic efficacy and speed of internalization in three CTCL cell lines, namely HuT-78, HH and MJ. In addition, the modulating effect of curcumin on selected proteins involved in the proliferation and progression of the disease was determined. The results indicated the superiority of the Pluronic®P-123/F-127 micellar curcumin over the standard ethanol solution in terms of cellular internalization efficiency as determined by spectrophotometric analysis. Notably, the presence of commonly used media components, such as phenol red, may interfere when interpreting the cytotoxicity of curcumin, due to their overlapping absorbance peaks. Therefore, it was concluded that phenol red-free media are superior over media with phenol red in order to correctly measure the cytotoxic efficacy and cell penetration of curcumin. Depending on the cell line, the IC50 values of micellar curcumin varied from 29.76 to 1.24 µΜ, with HH cells demonstrating the highest sensitivity. This cell line had the lowest expression levels of the Wilms' tumor-1 transcription factor. Performing western blot analyses of treated and untreated CTCL cells, selective signal transduction changes were recorded for the first time, thus making curcumin nano-formulation an attractive and prospective option with therapeutic relevance for CTCL as a rare orphan disease.

Dual SMO/BRAF Inhibition by Flavonolignans from Silybum marianum †.

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

Gypsophila saponins enhance the cytotoxicity of etoposide in HD-MY-Z lymphoma cells.

Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3,28-Bidesmosides (GOTCAB) are accumulated in Gypsophila L. roots. In the study we aimed at investigating the possible synergistic effects of Gypsophila trichotoma GOTCABs and cytostatic etoposide towards the Hodgkin lymphoma cell line HD-MY-Z. The combination effects with etoposide were evaluated using the symbolic mathematical software MAPLE. Liquid chromatography-mass spectrometry allowed the identification or tentative assignment of 28 core GOTCAB structures together with 6 monodesmosides in the root extract. Tested gypsogenin-based saponins possessed C-28 ester-bonded chain substituted with acetyl, cis/trans methoxycinnamoyl and both acetyl and sulfate groups. No cytotoxic effect was observed up to 20 µg/mL on normal mice fibroblasts (CCL-1 cell line) and lymphoma cells. Etoposide alone exerted IC50 93 µg/mL. In the presence of acetylated saponins (20 µg/mL), a strong synergism (Fa = 0.8, CI = 0.1) was observed with IC50 11 µg/mL. The combination induced apoptosis witnessed by caspase activation, elevated levels of cytosolic mono- and oligonucleosomes, and nuclear fragmentation together with discernible increase in ROS generation. The results emphasize the arabinose in the C-3 chain and acetylation pattern of the C-28 chain of the saponins as important structural features for cytotoxicity enhancing activity. Triterpenoid saponins are a valuable tool to improve the efficacy of cytostatics.

Complex mathematical analysis of photobioreactor system.

Modeling as a tool solves extremely difficult tasks in life sciences. Recently, schemes of culturing of microalgae have received special attention because of its unique features and possible uses in many industrial applications for renewable energy production and high value products isolation. The goal of this review is to present the use of system analysis theory applied to microalgae culturing modeling and process development. The review mainly focuses on the modeling of the key steps of autotrophic growth under the integral biorefinery concept of the microalgae biomass. The system approach follows systematically a procedure showing the difficulties by modeling of sub-systems. The development of microalgae kinetics and computational fluid dynamics (CFD) studies were analyzed in details as sub-systems in advanced design of photobioreactor (PBR). This review logically follows the trends of the modeling procedure and clarifies how this approach may save time and money during the research efforts. The result of this work is a successful development of a complex PBR mathematical analysis in the frame of the integral biorefinery concept.

Alkylphospholipids are Signal Transduction Modulators with Potential for Anticancer Therapy.

BACKGROUND: Alkylphospholipids (APLs) are synthetically derived from cell membrane components, which they target and thus modify cellular signalling and cause diverse effects. This study reviews the mechanism of action of anticancer, antiprotozoal, antibacterial and antiviral activities of ALPs, as well as their clinical use. METHODS: A literature search was used as the basis of this review. RESULTS: ALPs target lipid rafts and alter phospholipase D and C signalling cascades, which in turn will modulate the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK pathways. By feedback coupling, the SAPK/JNK signalling chain is also affected. These changes lead to a G2/M phase cell cycle arrest and subsequently induce programmed cell death. The available knowledge on inhibition of AKT phosphorylation, mTOR phosphorylation and Raf down-regulation renders ALPs as attractive candidates for modern medical treatment, which is based on individualized diagnosis and therapy. Corresponding to their unusual profile of activities, their side effects result from cholinomimetic activity mainly and focus on the gastrointestinal tract. These aspects together with their bone marrow sparing features render APCs well suited for modern combination therapy. Although the clinical success has been limited in cancer diseases so far, the use of miltefosine against leishmaniosis is leading the way to better understanding their optimized use. CONCLUSION: Recent synthetic programs generate congeners with the increased therapeutic ratio, liposomal formulations, as well as diapeutic (or theranostic) derivatives with optimized properties. It is anticipated that these innovative modifications will pave the way for the further successful development of ALPs.

Migratory birds along the Mediterranean - Black Sea Flyway as carriers of zoonotic pathogens.

At the crossroad between Europe, Asia, and Africa, Bulgaria is part of the Mediterranean - Black Sea Flyway (MBSF) used by millions of migratory birds. In this study, bird species migrating through Bulgaria were investigated as carriers of zoonotic pathogens. In total, 706 birds belonging to 46 species were checked for the presence of various bacterial pathogens (Campylobacter, Yersinia, Salmonella, Listeria, Escherichia coli, Staphylococcus aureus, Francisella tularensis, Coxiella burnetii, Borrelia burgdorferi, and Brucella spp.). From 673 birds we investigated fecal samples, from the remaining 33, blood samples. We detected Campylobacter 16S rDNA gene in 1.3% of birds, but none were of pathogenic Campylobacter jejuni and Campylobacter coli species. Escherichia coli 16S rDNA gene was found in 8.8% of the birds. Out of 34 birds that transported Yersinia enterocolitica strains (5.05%), only 1 carried a pathogenic isolate. Three birds (0.4%) were carriers of nonpathogenic Salmonella strains. Four avian samples (0.6%) were positive for Listeria monocytogenes and 1 (0.15%) was positive for Brucella spp. None of the birds tested carried the tick-borne pathogens C. burnetii or B. burgdorferi sensu lato. Antibiotic-resistant strains were detected, suggesting that migratory birds could be reservoirs and spreaders of bacterial pathogens as well as antibiotic resistance genes.

Antimicrobial and antioxidant potential of different solvent extracts of the medicinal plant Geum urbanum L.

Many Geum species are known to be rich in biologically active compounds and therefore could be a source of new natural products with pharmacological potential. The medicinal plant Geum urbanum L. is widespread in Bulgaria and has been used in folk medicine. In the present study, the methanol extracts of the roots and aerial parts of G. urbanum and their fractions (petroleum ether, ethyl acetate and n-butanol) were investigated for antibacterial and radical scavenging activity. The ethyl acetate and n-butanol fractions inhibited the growth of Gram-positive pathogenic and opportunistic bacteria from the genus Staphylococcus (MIC EtOAc: 0.078 mg/ml aerial and 0.156 mg/ml roots; MIC n-BuOH: 0.156 mg/ml aerial and 1.25 mg/ml roots) and the species Bacillus cereus stronger than the other extracts and fractions tested (MIC EtOAc: 0.078 mg/ml aerial and 0.156 mg/ml roots; MIC n-BuOH: 0.156 mg/ml aerial and 0.078 mg/ml roots), and showed corresponding radical scavenging activity (EtOAc: EC50 1.5 µg/ml aerial, 0.8 µg/ml roots; n-BuOH: 4.5 µg/ml aerial; 3.7 µg/ml roots). Additionally, their total phenolic content was quantified (% of dry EtOAc fractions of roots 61%, of arial parts 32%; of dry n-BuOH fractions of roots 16%, of arial parts 13%). Seven compounds were isolated and identified spectroscopically from the ethyl acetate extract. Two acetylated ellagic acid rhamnosides were found for the first time in the genus Geum and three others, tormentic acid, niga-ichigoside F1, and 3,3'-di-O-methylellagic acid-4-O-ß-D-glucopyranoside, were newly detected for the species G. urbanum. Our results reveal that G. urbanum L. is a perspective medicinal plant and deserves further, more detailed studies.

HPLC-UV and LC-MS Analyses of Acylquinic Acids in Geigeria alata (DC) Oliv. & Hiern. and their Contribution to Antioxidant and Antimicrobial Capacity.

INTRODUCTION: Geigeria alata is a traditional plant used in Sudanese folk medicine for treatment of diabetes, cough, epilepsy and intestinal complaints. OBJECTIVE: To analyze phenolic acids in Geigeria alata roots and leaves and to evaluate their antioxidant and antimicrobial activities. METHODOLOGY: Phenolic acids in the aqueous-methanol extracts were identified by LC-MS. Major compounds were isolated using low-pressure liquid chromatography. The quantitative analysis of phenolic acids was performed by a validated HPLC-UV method with limits of detection ranging from 0.04 to 0.57 µg/mL. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazine-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods were used for antioxidant activity evaluation. In addition, the minimal inhibitory concentration and the minimal bactericidal concentration against a panel of pathogenic bacteria and fungi were determined by the broth microdilution test. RESULTS: For the first time protocatechuic, caffeic, p-coumaroylquinic, caffeoylsinapoylquinic, caffeoylferuloylquinic, three feruloylquinic, six caffeoylquinic acids, and a caffeic acid hexoside were detected in Geigeria alata roots by LC-MS. HPLC-UV analyses showed that 3,5-dicaffeoylquinic acid (25.96 ± 2.08 mg/g dry weight (DW)) was the most abundant phenolic acid in roots, while 4,5-dicaffeoylquinic acid (8.99 ± 0.56 mg/g DW) was the main compound present in leaves. 3,5-Dicaffeoylquinic acid demonstrated stronger radical scavenging activity and reducing power compared with the crude extracts and the positive control 5-caffeoylquinic acid. 3,4,5-Tricaffeoylquinic acid revealed the highest antibacterial potential against the penicillin sensitive and resistant Staphylococcus aureus strains, as well as methicillin-resistant S. aureus. CONCLUSION: The caffeoylquinic acids content of up to 6.22% in Geigeria alata roots establishes this species as a new source rich in these bioactive molecules. Copyright © 2016 John Wiley & Sons, Ltd.

Reduced expression of the retinoblastoma protein shows that the related signaling pathway is essential for mediating the antineoplastic activity of erufosine.

Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by erufosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.

Erufosine suppresses breast cancer in vitro and in vivo for its activity on PI3K, c-Raf and Akt proteins.

PURPOSE: This study investigated the antineoplastic effect of the membrane active alkylphosphocholine erufosine in breast carcinoma models in vitro and in vivo and determined its influence on the PI3K/Akt and Ras/Raf/MAPK signaling pathways. METHODS: The antiproliferative effect of erufosine in vitro was determined by the MTT dye reduction assay, and the antineoplastic efficacy on tumor growth was investigated by relating the mean total tumor volumes of treated and control rats. Immunoblot analysis was used for detecting changes in the expression level of the signal molecules p-PI3K (p-p85), p-Akt at Thr 308 and p-cRaf. RESULTS: Based on their IC(50) (40 µM, respectively), the breast carcinoma cell lines MCF-7 and MDA-MB 231, which are estrogen receptor positive and negative, respectively, were equally sensitive to erufosine. In addition, erufosine caused dose-dependent decreases in the phosphorylation of PI3K (p85), Akt (PKB) at Thr 308 and cRaf in both cell lines. Moreover, administration of erufosine to rats bearing autochthonous methylnitrosourea-induced rat mammary carcinomas caused a significant dose-related tumor remission by more than 85 % (p < 0.05), which was well tolerated, as evidenced by a body weight loss of maximally 7 % and reduced tumor-related mortality (2 of 35 instead of 6 of 18 controls, p < 0.002). CONCLUSIONS: The results clearly indicate that erufosine possesses high antineoplastic activity not only in human breast cancer cell lines in vitro but also in rat mammary carcinoma in vivo. In addition, it can be derived that the mechanism of action of erufosine involves influence on both, PI3K/Akt and Ras/Raf/MAPK signaling pathways.

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.