Effect of Flavonols of Aronia melanocarpa Fruits on Morphofunctional State of Immunocompetent Organs of Rats under Cyclophosphamide-Induced Immunosuppression.

Aronia melanocarpa berries contain many compounds with potential benefits for human health. The food flavonoids quercetin and rutin, found in significant amounts in the fruits of A. melanocarpa, are known to have favourable effects on animal and human organisms. However, data on the effect of flavonols isolated from black chokeberry on immune functions during immunosuppression are not available in the literature. Thus, the aim of this study was to evaluate the effect of flavonol fraction isolated from A. melanocarpa fruits, in comparison with pure quercetin and rutin substances, on the dysfunctional state of rat thymus and spleen in immunodeficiency. The study was performed on Wistar rats. The animals were orally administered solutions of the investigated substances for 7 days: water, a mixture of quercetin and rutin and flavonol fraction of A. melanocarpa. For induction of immunosuppression, the animals were injected once intraperitoneally with cyclophosphamide. Substance administration was then continued for another 7 days. The results showed that under the influence of flavonols, there was a decrease in cyclophosphamide-mediated reaction of lipid peroxidation enhancement and stimulation of proliferation of lymphocytes of thymus and spleen in rats. At that, the effect of the flavonol fraction of aronia was more pronounced.

Synthesis, cytotoxicity and antioxidant activity of new conjugates of N-acetyl-d-glucosamine with α-aminophosphonates.

A series of the first conjugates of N-acetyl-d-glucosamine with α-aminophosphonates was synthesized using the Kabachnik-Fields reaction, the Pudovik reaction, a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and evaluated for the in vitro cytotoxicity against human cancer cell lines M - HeLa, HuTu-80, A549, PANC-1, MCF-7, T98G and normal lung fibroblast cells WI-38. The tested conjugates, with exception of compound 21b, considered as a lead compound, were either inactive against the used cancer cells or showed moderate cytotoxicity in the range of IC50 values 33-80 µM. The lead compound 21b, being non cytotoxic against normal human cells WI-38 (IC50 = 90 µM), demonstrated good activity (IC50 = 17 µM) against breast adenocarcinoma cells (MCF-7) which to be 1.5 times higher than the activity of the used reference anticancer drug tamoxifen (IC50 = 25.0 µM). A flexible receptor molecular docking simulation showed that the cytotoxicity of the synthesized conjugates of N-acetyl-d-glucosamine with α-aminophosphonates against breast adenocarcinoma MCF-7 cell line is due to their ability to inhibit EGFR kinase domain. In addition, it was found that conjugates 22a and 22b demonstrated antioxidant activity that was not typical for α-aminophosphonates.

Novel Mitochondria-Targeted Amphiphilic Aminophosphonium Salts and Lipids Nanoparticles: Synthesis, Antitumor Activity and Toxicity.

The creation of mitochondria-targeted vector systems is a new tool for the treatment of socially significant diseases. Phosphonium groups provide targeted delivery of drugs through biological barriers to organelles. For this purpose, a new class of alkyl(diethylAmino)(Phenyl) Phosphonium halides (APPs) containing one, two, or three diethylamino groups was obtained by the reaction of alkyl iodides (bromides) with (diethylamino)(phenyl)phosphines under mild conditions (20 °C) and high yields (93-98%). The structure of APP was established by NMR and XRD. A high in vitro cytotoxicity of APPs against M-HeLa, HuTu 80, PC3, DU-145, PANC-1, and MCF-7 lines was found. The selectivity index is in the range of 0.06-4.0 µM (SI 17-277) for the most active APPs. The effect of APPs on cancer cells is characterized by hyperproduction of ROS and depolarization of the mitochondrial membrane. APPs induce apoptosis, proceeding along the mitochondrial pathway. Incorporation of APPs into lipid systems (liposomes and solid lipid nanoparticles) improves cytotoxicity toward tumor cells and decrease toxicity against normal cell lines. The IC50s of lipid systems are lower than for the reference drug DOX, with a high SI (30-56) toward MCF-7 and DU-145. APPs exhibit high selective activity against Gram-positive bacteria S. aureus 209P and B. segeus 8035, including methicillin-resistant S. aureus (MRSA-1, MRSA-2), comparable to the activity of the fluoroquinolone antibiotic norfloxacin. A moderate in vivo toxicity in CD-1 mice was established for the lead APP.

Aronia melanocarpa Flavonol Extract-Antiradical and Immunomodulating Activities Analysis.

The study of Aronia melanocarpa's (A. melanocarpa) biological activity is focused on obtaining the crude extract and separation of the flavonol compounds. The extraction and fractionation of A. melanocarpa fruits, followed by quantitative analysis, were accomplished using high-performance liquid chromatography and Darco G-60 filtering. This approach enabled the quantification of flavonoids within each fraction. The antioxidative, immunomodulating activities and cytotoxicity with respect to the lymphoblast cell line RPMI-1788 were studied. The flavonol extract of A. melanocarpa has been shown to have a high capacity to neutralize free DPPH and AAPH radicals in vitro. It also caused an accelerated 'respiratory burst' formation of neutrophils and an increase in the metabolic reserves of cells in rats exposed to cyclophosphamide. The reference solution (an equivalent quercetin-rutin blend) contributed to a decrease in lipid peroxidation, intensifying phagocytosis processes. The studied compounds demonstrated their low influence on the leukocyte blood profile in animals.

Mitochondria-Targeted Delivery Strategy of Dual-Loaded Liposomes for Alzheimer's Disease Therapy.

Liposomes modified with tetradecyltriphenylphosphonium bromide with dual loading of α-tocopherol and donepezil hydrochloride were successfully designed for intranasal administration. Physicochemical characteristics of cationic liposomes such as the hydrodynamic diameter, zeta potential, and polydispersity index were within the range from 105 to 115 nm, from +10 to +23 mV, and from 0.1 to 0.2, respectively. In vitro release curves of donepezil hydrochloride were analyzed using the Korsmeyer-Peppas, Higuchi, First-Order, and Zero-Order kinetic models. Nanocontainers modified with cationic surfactant statistically better penetrate into the mitochondria of rat motoneurons. Imaging of rat brain slices revealed the penetration of nanocarriers into the brain. Experiments on transgenic mice with an Alzheimer's disease model (APP/PS1) demonstrated that the intranasal administration of liposomes within 21 days resulted in enhanced learning abilities and a reduction in the formation rate of Aß plaques in the entorhinal cortex and hippocampus of the brain.

Antioxidative and Immunomodulating Properties of Aronia melanocarpa Extract Rich in Anthocyanins.

The fruits of Aronia melanocarpa are well known due to their high anthocyanin content that may be effective in preventing certain health disorders arising from oxidative stress. Various polyphenolic compounds such as anthocyanins and flavonoids are responsible for the multiple effects of chokeberry. The aim of this study was to determine in vitro how active the black chokeberry anthocyanins are in scavenging radicals and to evaluate in vivo their immunomodulating capacity. Using the method of column chromatography, we extracted the anthocyanins of black chokeberries, i.e., cyanidin-3-O-galactoside with a purity of over 93.7%. Using HPLC and spectrophotometric analysis, the flavonoid content was determined. Following the analysis of the tests with AAPH and DPPH, the chokeberry cyanidin-3-O-galactoside was found much better than individual anthocyanins in regard to antioxidant capacity. The range of concentrations was revealed, showing the protective effect of anthocyanins on the RPMI-1788 cell culture against cyclophosphamide, as well as against osmotic and peroxide hemolysis. An immunomodulating effect on the functional activity of phagocytes was revealed in vivo as a result of oral administration of chokeberry cyanidin-3-O-galactoside and a mixture composed of cyanidin-3-O-glucoside and cyanidin-3-O-galactoside standards. Consequently, anthocyanins, in particular cyanidin-3-O-galactoside, play an important role, demonstrating immunomodulating effects when chokeberries are consumed.

Supramolecular Tools to Improve Wound Healing and Antioxidant Properties of Abietic Acid: Biocompatible Microemulsions and Emulgels.

Abietic acid, a naturally occurring fir resin compound, that exhibits anti-inflammatory and wound-healing properties, was formulated into biocompatible emulgels based on stable microemulsions with the addition of a carbamate-containing surfactant and Carbopol® 940 gel. Various microemulsion and emulgel formulations were tested for antioxidant and wound-healing properties. The chemiluminescence method has shown that all compositions containing abietic acid have a high antioxidant activity. Using Strat-M® skin-modelling membrane, it was found out that emulgels significantly prolong the release of abietic acid. On Wistar rats, it was shown that microemulsions and emulgels containing 0.5% wt. of abietic acid promote the rapid healing of an incised wound and twofold tissue reinforcement compared to the untreated group, as documented by tensiometric wound suture-rupture assay. The high healing-efficiency is associated with a combination of antibacterial activity of the formulation components and the anti-inflammatory action of abietic acid.

Effects of Phenotypic Variation on Biological Properties of Endophytic Bacteria Bacillus mojavensis PS17.

The use of microorganism-based products in agricultural practices is gaining more interest as an alternative to chemical methods due to their non-toxic bactericidal and fungicidal properties. Various factors influence the efficacy of the microorganisms used as biological control agents in infield conditions as compared to laboratory conditions due to ecological and physiological aspects. Abiotic factors have been shown to trigger phase variations in bacterial microorganisms as a mechanism for adapting to hostile environments. In this study, we investigated the stability of the morphotype and the effects of phenotypic variation on the biological properties of Bacillus mojavensis strain PS17. B. mojavensis PS17 generated two variants (opaque and translucent) that were given the names morphotype I and II, respectively. The partial sequence of the 16S rRNA gene revealed that both morphotypes belonged to B. mojavensis. BOX and ERIC fingerprinting PCR also showed the same DNA profiles in both morphotypes. The characteristics of morphotype I did not differ from the original strain, while morphotype II showed a lower hydrolytic enzyme activity, phytohormone production, and antagonistic ability against phytopathogenic fungi. Both morphotypes demonstrated endophytic ability in tomato plants. A low growth rate of the strain PS17(II) in a minimal medium was observed in comparison to the PS17(I) strain. Furthermore, the capacity for biocontrol of B. mojavensis PS17(II) was not effective in the suppression of root rot disease in the tomato plants caused by Fusarium oxysporum f. sp. radices-lycopersici stain ZUM2407, compared to B. mojavensis PS17(I), whose inhibition was almost 47.9 ± 1.03% effective.

Cellular and Molecular Gradients in the Ventral Horns With Increasing Distance From the Injury Site After Spinal Cord Contusion.

To identify cellular and molecular gradients following spinal cord injury (SCI), a rat contusion model of severe SCI was used to investigate the expression of NG2 and molecules that identify astrocytes and axons of the ventral horns (VH) at different distances on 7 and 30 days post-injury (dpi). A gradient of expression of NG2+/Olig2+ cells was determined, with the highest concentrations focused close to the injury site. A decrease in NG2 mean intensity correlates with a decrease in the number of NG2+ cells more distally. Immunoelectron microscopy subsequently revealed the presence of NG2 in connection with the membrane and within the cytoplasm of NG2+ glial cells and in large amounts within myelin membranes. Analysis of the astrocyte marker GFAP showed increased expression local to injury site from 7 dpi, this increase in expression spread more distally from the injury site by 30 dpi. Paradoxically, astrocyte perisynaptic processes marker GLT-1 was only increased in expression in areas remote from the epicenter, which was traced both at 7 and 30 dpi. Confocal microscopy showed a significant decrease in the number of 5-HT+ axons at a distance from the epicenter in the caudal direction, which is consistent with a decrease in ß3-tubulin in these areas. The results indicate significant cellular and molecular reactions not only in the area of the gray matter damage but also in adjacent and remote areas, which is important for assessing the possibility of long-distance axonal growth.

Microemulsions and nanoemulsions modified with cationic surfactants for improving the solubility and therapeutic efficacy of loaded drug indomethacin.

In this work, a noncovalent strategy was successfully used to modify colloidal stability andin vitroandin vivoefficacy of two amphiphilic formulations of the anti-inflammatory drug indomethacin. Namely, nanoemulsions and microemulsions based on oleic acid and nonionic surfactants have been produced and compared. The influence of cationic surfactants cetyltrimethylammonium bromide and its carbamate bearing analogue on the size characteristics, stability and ability to provide prolonged action of loaded drug indomethacin has been evaluated. Adding the positively charged molecules in the surface layer of nanoemulsions and microemulsions has shown the stability increase along with maintaining the size characteristics and homogeneity in time. Moreover, the carbamate modified analogue demonstrated beneficial behavior. Indomethacin loaded in microemulsions and nanoemulsions showed prolonged-release (10%-15% release for 5 h) compared to a free drug (complete release for 5 h). The rate of release of indomethacin from nanoemulsions was slightly higher than from microemulsions and insignificantly decreased with an increase in the concentration of the cationic surfactant. For carbamate surfactant nanocarrier loaded with fluorescence probe Nile Red, the ability to penetrate into the cell was supported by flow cytometry study and visualized by fluorescence microscopy.In vitrotests on anti-inflammatory activity of the systems demonstrated that the blood cell membrane stabilization increased in the case of modified microemulsion. The anti-inflammatory activity of the encapsulated drug was tested in rats using a carrageenan-induced edema model. Nanoemulsions without cationic surfactants appeared more efficient compared to microemulsions. Indomethacin emulsion formulations with carbamate surfactant added showed slower carrageenan-induced edema progression compared to unmodified compositions. Meanwhile, the edema completely disappeared upon treatment with emulsion loaded indomethacin after 4 h in the case of microemulsions versus 5 h in the case of nanoemulsions.

Radical Scavenging Actions and Immunomodulatory Activity of Aronia melanocarpa Propylene Glycol Extracts.

Researchers are attracted to the wide-ranging, useful components in Aronia melanocarpa berries. They are searching for the most effective ways to extract the active substances that can enhance the body's protective properties. The current study presents detailed information about the extracts from A. melanocarpa fruits frozen and dried under mild conditions and their chemical composition. In Wistar rats with induced immunosuppression, the effect of chokeberry fruit extracts on the leukocyte formula, phagocytic activity, and cytokine system was studied. It was shown that the A. melanocarpa frozen fruit extract contains more anthocyanins, sugars, and ascorbic acid, and has a more pronounced antioxidant activity determined by the ability to bind APPH-radicals. Moreover, the extract showed membrane-protective and cytoprotective properties against RPMI-1788 cell line. The extract from dried raw material shows a higher antioxidant activity due to the ability to bind DPPH-radicals. It was revealed that extracts from A. melanocarpa fruits promote rapid immune system recovery in rats, normalize the leukocyte count, and improve monocyte and neutrophil phagocytic indicators. Research on the cytokine profile revealed that the anti-inflammatory properties in A. melanocarpa extracts were more pronounced in dried extracts. For several cytokines, a normalization of quantity was noted.

Conjugate of pyrimidine derivative, the drug xymedon with succinic acid protects liver cells.

The aim of the study was to investigate the hepatoprotective properties of the conjugate of the xymedon drug substance with succinic acid (1a). The study presents an in vitro comparative evaluation of the cytotoxicity and cytoprotective properties of 1a and succinic acid on a cell line of normal human hepatocytes Chang Liver, and in vivo investigation of the ability of 1a to restore liver from the toxic damage caused by CCl4 in Wistar rats. It was shown that the cytotoxicity of 1a was 19.9 ± 0.8 mmol/L, and that of succinic acid was 14.1 ± 0.2 mmol/L. Against the background of d-galactosamine exposure, the cytoprotective effect of 1a was found to be superior to that of succinic acid. It was shown that 1a caused a significant reduction in necrotic and steatosis changes in the liver and restoration of biochemical markers of cytolysis, as well as bilirubin metabolism and synthetic liver function.

Bi-functional sterically hindered phenol lipid-based delivery systems as potential multi-target agents against Alzheimer's disease via an intranasal route.

New lipid-based nanomaterials and multi-target directed ligands (MTDLs) based on sterically hindered phenol, containing a quaternary ammonium moiety (SHP-s-R, with s = 2,3) of varying hydrophobicity (R = CH2Ph and CnH2n+1, with n = 8, 10, 12, 16), have been prepared as potential drugs against Alzheimer's disease (AD). SHP-s-R are inhibitors of human cholinesterases with antioxidant properties. The inhibitory potency of SHP-s-R and selectivity ratio of cholinesterase inhibition were found to significantly depend on the length of the methylene spacer (s) and alkyl chain length. The compound SHP-2-16 showed the best IC50 for human AChE and the highest selectivity, being 30-fold more potent than for human BChE. Molecular modeling of SHP-2-16 binding to human AChE suggests that this compound is a dual binding site inhibitor that interacts with both the peripheral anionic site and catalytic active site. The relationship between self-assembly parameters (CMC, solubilization capacity, aggregation number), antioxidant activity and a toxicological parameter (hemolytic action on human red blood cells) was investigated. Two sterically hindered phenols (SHP-2-Bn and SHP-2-R) were loaded into L-α-phosphatidylcholine (PC) nanoparticles by varying the SHP alkyl chain length. For the brain AChE inhibition assay, PC/SHP-2-Bn/SHP-2-16 nanoparticles were administered to rats intranasally at a dose of 8 mg kg-1. The Morris water maze experiment showed that scopolamine-induced AD-like dementia in rats treated with PC/SHP-2-Bn/SHP-2-16 nanoparticles was significantly reduced. This is the first example of cationic SHP-phospholipid nanoparticles for inhibition of brain cholinesterases realized by the use of intranasal administration. This route has promising potential for the treatment of AD.

New 2,6-diaminopyridines containing a sterically hindered benzylphosphonate moiety in the aromatic core as potential antioxidant and anti-cancer drugs.

A series of 2,6-diaminopyridines was synthesized for the first time, containing phosphoryl sterically hindered phenolic fragments in the aromatic core. The antioxidant activity of these compounds was investigated, 2,6-diaminopyridine derivatives were shown to exhibit higher activity in comparison with their structural analogues. For dialkyl/diphenyl [(3,5-di-tert-butyl-4-hydroxyphenyl) (2,6-diaminopyridin-3-yl) methyl] phosphonates, their structural analogues based on meta-phenylenediamine, phosphorus-containing sterically hindered phenols and the corresponding cyclohexadienones cytotoxicity against tumor lines of epithelioid carcinoma of the cervix uteri (M-Hela) and breast adenocarcinoma (MCF-7) has been studied in vitro, as well as on normal human Chang liver cell lines. Diphenyl [(3,5-di-tert-butyl-4-hydroxyphenyl) (2,6-diaminopyridin-3-yl) methyl] phosphonate was shown to be the most active against the epithelioid line M-Hela - IC50 comprises 7.4 µM. It was shown that apoptosis induced by the lead compound proceeds along the internal pathway of caspase-9 activation. It was established that all studied compounds do not possess hemolytic activity.

Design, synthesis, and biological activity of novel ammonium salts containing sterically hindered phenolic fragment and phosphoryl group.

Here, we present an approach to novel "hybrid" biologically active compounds based on a combination of sterically hindered phenol and ammonium pharmacophores in a single molecule. The novel target ammonium salts were obtained by the reaction of 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-(2-(dimethylamino)alkyl)propanamide with aliphatic bromides or by the reaction of phosphorylated methylenequinones with diamines followed by alkylation with organic bromides. A series of twenty-three novel multifunctional ammonium salts that contain a sterically hindered phenolic fragment were assessed for antimicrobial, cytotoxic and antioxidant activity. The compounds exhibited antimicrobial activity against Staphylococcus aureus ATCC 209p, Bacillus cereus ATCC 8035, Escherichia coli CDC F-50, Pseudomonas aeruginosa ATCC 9027, Aspergillus niger BKMF-1119, Trichophyton mentagrophytes var. gypseum 1773, and Candida albicans 855-653 in the concentration range of 442-0.70 µM. The maximum activity of an ammonium salt among all the types of structure was observed in cases in which a decyl radical was present on the onium nitrogen atom. The most active compounds exhibited antioxidant activity at levels of 0.25 and 0.50 mM and did not display cytotoxic properties towards WI-38 (human embryonic lung cells) and Chang liver (human liver cells) cell lines in the concentration range of 0.70-11.3 µM.