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IQ-1 (11 H -indeno[1,2- b ]quinoxalin-11-one oxime) is a specific c-Jun N-terminal kinase (JNK) inhibitor with anticancer and neuro- and cardioprotective properties. Because aryloxime derivatives undergo cytochrome P450-catalyzed oxidation to nitric oxide (NO) and ketones in liver microsomes, NO formation may be an additional mechanism of IQ-1 pharmacological action. In the present study, electron paramagnetic resonance (EPR) of the Fe 2+ complex with diethyldithiocarbamate (DETC) as a spin trap and hemoglobin (Hb) was used to detect NO formation from IQ-1 in the liver and blood of rats, respectively, after IQ-1 intraperitoneal administration (50 mg/kg). Introducing the spin trap and IQ-1 led to signal characteristics of the complex (DETC) 2 -Fe 2+ -NO in rat liver. Similarly, the introduction of the spin trap components and IQ-1 resulted in an increase in the Hb-NO signal for both the R- and the T-conformers in blood samples. The density functional theory (DFT) calculations were in accordance with the experimental data and indicated that the NO formation of IQ-1 through the action of superoxide anion radical is thermodynamically favorable. We conclude that the administration of IQ-1 releases NO during its oxidoreductive bioconversion in vivo .

Chronic inflammation contributes to a number of diseases. Therefore, control of the inflammatory response is an important therapeutic goal. To identify novel anti-inflammatory compounds, we synthesized and screened a library of 80 pyrazolo[1,5- a ]quinazoline compounds and related derivatives. Screening of these compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP-1Blue monocytic cells identified 13 compounds with anti-inflammatory activity (IC 50 < 50 µM) in a cell-based test system, with two of the most potent being compounds 13i (5-[(4-sulfamoylbenzyl)oxy]pyrazolo[1,5- a ]quinazoline-3-carboxamide) and 16 (5-[(4-(methylsulfinyl)benzyloxy]pyrazolo[1,5- a ]quinazoline-3-carboxamide). Pharmacophore mapping of potential targets predicted that 13i and 16 may be ligands for three mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2), p38α, and c -Jun N -terminal kinase 3 (JNK3). Indeed, molecular modeling supported that these compounds could effectively bind to ERK2, p38α, and JNK3, with the highest complementarity to JNK3. The key residues of JNK3 important for this binding were identified. Moreover, compounds 13i and 16 exhibited micromolar binding affinities for JNK1, JNK2, and JNK3. Thus, our results demonstrate the potential for developing lead anti-inflammatory drugs based on the pyrazolo[1,5- a ]quinazoline and related scaffolds that are targeted toward MAPKs.

Echinacea purpurea (L.) Moench is a medicinal plant commonly used for the treatment of upper respiratory tract infections, the common cold, sore throat, migraine, colic, stomach cramps, and toothaches and the promotion of wound healing. Based on the known pharmacological properties of essential oils (EOs), we hypothesized that E. purpurea EOs may contribute to these medicinal properties. In this work, EOs from the flowers of E. purpurea were steam-distilled and analyzed by gas chromatography-mass spectrometry (GC-MS), GC with flame-ionization detection (GC-FID), and chiral GC-MS. The EOs were also evaluated for in vitro antimicrobial and innate immunomodulatory activity. About 87 compounds were identified in five samples of the steam-distilled E. purpurea EO. The major components of the E. purpurea EO were germacrene D (42.0 ± 4.61%), α-phellandrene (10.09 ± 1.59%), β-caryophyllene (5.75 ± 1.72%), γ-curcumene (5.03 ± 1.96%), α-pinene (4.44 ± 1.78%), δ-cadinene (3.31 ± 0.61%), and β-pinene (2.43 ± 0.98%). Eleven chiral compounds were identified in the E. purpurea EO, including α-pinene, sabinene, β-pinene, α-phellandrene, limonene, β-phellandrene, α-copaene, β-elemene, β-caryophyllene, germacrene D, and δ-cadinene. Analysis of E. purpurea EO antimicrobial activity showed that they inhibited the growth of several bacterial species, although the EO did not seem to be effective for Staphylococcus aureus . The E. purpurea EO and its major components induced intracellular calcium mobilization in human neutrophils. Additionally, pretreatment of human neutrophils with the E. purpurea EO or (+)-δ-cadinene suppressed agonist-induced neutrophil calcium mobilization and chemotaxis. Moreover, pharmacophore mapping studies predicted two potential MAPK targets for (+)-δ-cadinene. Our results are consistent with previous reports on the innate immunomodulatory activities of β-caryophyllene, α-phellandrene, and germacrene D. Thus, this study identified δ-cadinene as a novel neutrophil agonist and suggests that δ-cadinene may contribute to the reported immunomodulatory activity of E. purpurea .

The genus Saussurea has been used in the preparation of therapies for a number of medical problems, yet not much is known about the therapeutic high-molecular-weight compounds present in extracts from these plants. Since polysaccharides are important in immune modulation, we investigated the chemical composition and immunomodulatory activity of Saussurea salicifolia L. and Saussurea frolovii Ledeb polysaccharides. Water-soluble polysaccharides from the aerial parts of these plants were extracted using water at pHs of 2 and 6 and subsequently precipitated in ethanol to obtain fractions SSP2 and SSP6 from S. salicifolia and fractions SSF2 and SSF6 from S. frolovii . The molecular weights of fractions SSP2, SSP6, SFP2, and SFP6 were estimated to be 143.7, 113.2, 75.3, and 64.3 kDa, respectively. The polysaccharides from S. frolovii contained xylose (67.1-71.7%) and glucose (28.3-32.9%), whereas the polysaccharides from S. frolovii contained xylose (63.1-76.7%), glucose (11.8-19.2%), galactose (4.7-8.3%), and rhamnose (6.8-9.4%). Fractions SSP2, SSP6, and SFP2 stimulated nitric oxide (NO) production by murine macrophages, and NO production induced by SSP2, SSP6, and SFP2 was not inhibited by polymyxin B treatment of the fractions, whereaspolymyxin B treatment diminished the effects of SFP6, suggesting that SFP6 could contain lipopolysaccharide (LPS). The LPS-free fractions SSP2, SSP6, and SFP2 had potent immunomodulatory activity, induced NO production, and activated transcription factors NF-κB/AP-1 in human monocytic THP-1 cells and cytokine production by human MonoMac-6 monocytic cells, including interleukin (IL)-1α, IL-1β, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF). These data suggest that at least part of the beneficial therapeutic effects reported for water extracts of the Saussurea species are due to the modulation of leukocyte functions by polysaccharides.

The c -Jun N-terminal kinase (JNK) family includes three proteins (JNK1-3) that regulate many physiological processes, including cell proliferation and differentiation, cell survival, and inflammation. Because of emerging data suggesting that JNK3 may play an important role in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease, as well as cancer pathogenesis, we sought to identify JNK inhibitors with increased selectivity for JNK3. A panel of 26 novel tryptanthrin-6-oxime analogs was synthesized and evaluated for JNK1-3 binding (K d ) and inhibition of cellular inflammatory responses. Compounds 4d (8-methoxyindolo[2,1- b ]quinazolin-6,12-dione oxime) and 4e (8-phenylindolo[2,1- b ]quinazolin-6,12-dione oxime) had high selectivity for JNK3 versus JNK1 and JNK2 and inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcriptional activity in THP-1Blue cells and interleukin-6 (IL-6) production by MonoMac-6 monocytic cells in the low micromolar range. Likewise, compounds 4d , 4e , and pan-JNK inhibitor 4h (9-methylindolo[2,1- b ]quinazolin-6,12-dione oxime) decreased LPS-induced c -Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. Molecular modeling suggested modes of binding interaction of these compounds in the JNK3 catalytic site that were in agreement with the experimental data on JNK3 binding. Our results demonstrate the potential for developing anti-inflammatory drugs based on these nitrogen-containing heterocyclic systems with selectivity for JNK3.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment due in part to a severe loss of cholinergic neurons in specific brain areas. AD is the most common type of dementia in the aging population. Although several acetylcholinesterase (AChE) inhibitors are currently available, their performance sometimes yields unexpected results. Thus, research is ongoing to find potentially therapeutic AChE inhibitory agents, both from natural and synthetic sources. Here, we synthesized 13 new lupinine triazole derivatives and evaluated them, along with 50 commercial lupinine-based esters of different carboxylic acids, for AChE inhibitory activity. The triazole derivative 15 [1 S ,9a R )-1-((4-(4-(benzyloxy)-3-methoxyphenyl)-1 H -1,2,3-triazol-1-yl)methyl)octahydro-2 H -quinolizine)] exhibited the most potent AChE inhibitory activity among all 63 lupinine derivatives, and kinetic analysis demonstrated that compound 15 was a mixed-type AChE inhibitor. Molecular docking studies were performed to visualize interaction between this triazole derivative and AChE. In addition, a structure-activity relationship (SAR) model developed using linear discriminant analysis (LDA) of 11 SwissADME descriptors from the 50 lupinine esters revealed 5 key physicochemical features that allowed us to distinguish active versus non-active compounds. Thus, this SAR model could be applied for design of more potent lupinine ester-based AChE inhibitors.

Grindelia squarrosa (Pursh) Dunal is used in traditional medicine for treating various diseases; however, little is known about the immunomodulatory activity of essential oils from this plant. Thus, we isolated essential oils from the flowers (GEO Fl ) and leaves (GEO Lv ) of G. squarrosa and evaluated the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of these essential oils revealed that the main components were α-pinene (24.7 and 23.2% in GEO Fl and GEO Lv , respectively), limonene (10.0 and 14.7%), borneol (23.4 and 16.6%), p -cymen-8-ol (6.1 and 5.8%), β-pinene (4.0 and 3.8%), bornyl acetate (3.0 and 5.1%), trans -pinocarveol (4.2 and 3.7%), spathulenol (3.0 and 2.0%), myrtenol (2.5 and 1.7%), and terpinolene (1.7 and 2.0%). Enantiomer analysis showed that α-pinene, β-pinene, and borneol were present primarily as (-)-enantiomers (100% enantiomeric excess (ee) for (-)-α-pinene and (-)-borneol in both GEO Fl and GEO Lv ; 82 and 78% ee for (-)-β-pinene in GEO Fl and GEO Lv ), while limonene was present primarily as the (+)-enantiomer (94 and 96 ee in GEO Fl and GEO Lv ). Grindelia essential oils activated human neutrophils, resulting in increased [Ca 2+ ] i (EC 50 = 22.3 µg/mL for GEO Fl and 19.4 µg/mL for GEO Lv ). In addition, one of the major enantiomeric components, (-)-borneol, activated human neutrophil [Ca 2+ ] i (EC 50 = 28.7 ± 2.6), whereas (+)-borneol was inactive. Since these treatments activated neutrophils, we also evaluated if they were able to down-regulate neutrophil responses to subsequent agonist activation and found that treatment with Grindelia essential oils inhibited activation of these cells by the N -formyl peptide receptor 1 (FPR1) agonist f MLF and the FPR2 agonist WKYMVM. Likewise, (-)-borneol inhibited FPR-agonist-induced Ca 2+ influx in neutrophils. Grindelia leaf and flower essential oils, as well as (-)-borneol, also inhibited f MLF-induced chemotaxis of human neutrophils (IC 50 = 4.1 ± 0.8 µg/mL, 5.0 ± 1.6 µg/mL, and 5.8 ± 1.4 µM, respectively). Thus, we identified (-)-borneol as a novel modulator of human neutrophil function.

Persistent inflammation contributes to a number of diseases; therefore, control of the inflammatory response is an important therapeutic goal. In an effort to identify novel anti-inflammatory compounds, we screened a library of pyridazinones and structurally related derivatives that were used previously to identify N-formyl peptide receptor (FPR) agonists. Screening of the compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP1-Blue monocytic cells identified 48 compounds with anti-inflammatory activity. Interestingly, 34 compounds were FPR agonists, whereas 14 inhibitors of LPS-induced NF-κB activity were not FPR agonists, indicating that they inhibited different signaling pathways. Further analysis of the most potent inhibitors showed that they also inhibited LPS-induced production of interleukin 6 (IL-6) by human MonoMac-6 monocytic cells, again verifying their anti-inflammatory properties. Structure-activity relationship (SAR) classification models based on atom pair descriptors and physicochemical ADME parameters were developed to achieve better insight into the relationships between chemical structures of the compounds and their biological activities, and we found that there was little correlation between FPR agonist activity and inhibition of LPS-induced NF-κB activity. Indeed, Cmpd43, a well-known pyrazolone-based FPR agonist, as well as FPR1 and FPR2 peptide agonists had no effect on the LPS-induced NF-κB activity in THP1-Blue cells. Thus, some FPR agonists reported to have anti-inflammatory activity may actually mediate their effects through FPR-independent pathways, as it is suggested by our results with this series of compounds. This could explain how treatment with some agonists known to be inflammatory (i.e., FPR1 agonists) could result in anti-inflammatory effects. Further research is clearly needed to define the molecular targets of pyridazinones and structurally related compounds with anti-inflammatory activity and to define their relationships (if any) to FPR signaling events.

Little is known about the immunomodulatory activity of essential oils isolated from Juniperus species. Thus, we isolated essential oils from the cones and leaves of eight juniper species found in Montana and in Kazakhstan, including J. horizontalis , J. scopolorum , J. communis , J. seravschanica , J. sabina , J. pseudosabina , J. pseudosabina subsp. turkestanica , and J. sibirica . We report here the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of the 16 samples of Juniper essential oils revealed similarities and differences between our analyses and those previously reported for essential oils from this species. Our studies represent the first analysis of essential oils isolated from the cones of four of these Juniper species. Several essential oil samples contained high levels of cedrol, which was fairly unique to three Juniper species from Kazakhstan. We found that these essential oils and pure (+)-cedrol induced intracellular Ca 2+ mobilization in human neutrophils. Furthermore, pretreatment of human neutrophils and N -formyl peptide receptor 1 and 2 (FPR1 and FPR2) transfected HL60 cells with these essential oils or (+)-cedrol inhibited agonist-induced Ca 2+ mobilization, suggesting these responses were desensitized by this pretreatment. In support of this conclusion, pretreatment with essential oils from J. seravschanica cones (containing 16.8% cedrol) or pure (+)-cedrol inhibited human neutrophil chemotaxis to N -formyl peptide. Finally, reverse pharmacophore mapping predicted several potential kinase targets for cedrol. Thus, our studies have identified cedrol as a novel neutrophil agonist that can desensitize cells to subsequent stimulation by N -formyl peptide.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint inflammation, cartilage damage and bone destruction. Although the pharmacological treatment of RA has evolved over the last few years, the new drugs have serious side effects and are very expensive. Thus, the research has been directed in recent years towards new possible targets. Among these targets, N-formyl peptide receptors (FPRs) are of particular interest. Recently, the mixed FPR1/FPR2 agonist Cpd43 , the FPR2 agonist AT-01-KG , and the pyridine derivative AMC3 have been shown to be effective in RA animal models. As an extension of this research, we report here a new series of pyridinone derivatives containing the (substituted)phenyl acetamide chain, which was found to be essential for activity, but with different substitutions at position 5 of the scaffold. The biological results were also supported by molecular modeling studies and additional pharmacological tests on AMC3 have been performed in a rat model of RA, by repeating the treatments of the animals with 10 mg/kg/day of compound by 1 week.

c-Jun N-terminal kinase (JNK) plays a central role in stress signaling pathways implicated in important pathological processes, including rheumatoid arthritis and ischemia-reperfusion injury. Therefore, inhibition of JNK is of interest for molecular targeted therapy to treat various diseases. We synthesized 13 derivatives of our reported JNK inhibitor 11 H -indeno[1,2- b ]quinoxalin-11-one oxime and evaluated their binding to the three JNK isoforms and their biological effects. Eight compounds exhibited submicromolar binding affinity for at least one JNK isoform. Most of these compounds also inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation and interleukin-6 (IL-6) production in human monocytic THP1-Blue cells and human MonoMac-6 cells, respectively. Selected compounds ( 4f and 4m ) also inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. We conclude that indenoquinoxaline-based oximes can serve as specific small-molecule modulators for mechanistic studies of JNKs, as well as potential leads for the development of anti-inflammatory drugs.

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REO Fl ) versus leaf (REO Lv ) essential oils revealed significant differences. REO Fl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REO Lv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REO Lv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca 2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N -formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca 2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca 2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca 2+ ] i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and ε-aminocaproyl substituents and analyzed their biological effects in vitro. Ten of the compounds ( 8 , 10 - 17 , and 21 ) increased intracellular calcium ([Ca 2+ ] i ) in human neutrophils, with the most potent being compound 15 ( N , N '- bis [2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could alter normal neutrophil [Ca 2+ ] i flux. Indeed, a number of these compounds (i.e., 8 , 10 - 17 , and 21 ) inhibited [Ca 2+ ] i flux in human neutrophils activated by N -formyl peptide ( f MLF). Some of these compounds also inhibited chemotactic peptide-induced [Ca 2+ ] i flux in HL60 cells transfected with N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate (PMA) and neutrophil chemotaxis toward f MLF, as both of these processes are highly dependent on regulated [Ca 2+ ] i flux. Quantum chemical calculations were performed on five structure-related diaza-crown ethers and their complexes with Ca 2+ , Na + , and K + to obtain a set of molecular electronic properties and to correlate these properties with biological activity. According to density-functional theory (DFT) modeling, Ca 2+ ions were more effectively bound by these compounds versus Na + and K + . The DFT-optimized structures of the ligand-Ca 2+ complexes and quantitative structure-activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the N , N '-diacylated diaza-crown ethers participated in cation binding and could play an important role in Ca 2+ transfer. Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore mechanism of biological action of aza-crown ethers.

Little is known about the pharmacological activity of Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEO Fl ) and leaf (MEO Lv ) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p -cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEO Fl and MEO Lv included carvacrol and thymol. Both MEO Fl and MEO Lv stimulated a transient increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEO Lv being much more effective than MEO Fl . Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca 2+ ] i induced by MEO Fl /MEO Lv , carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels.

Organosulfur compounds are bioactive components of garlic essential oil (EO), mustard oil, Ferula EOs, asafoetida, and other plant and food extracts. Traditionally, garlic ( Allium sativum ) is used to boost the immune system; however, the mechanisms involved in the putative immunomodulatory effects of garlic are unknown. We investigated the effects of garlic EO and 22 organosulfur compounds on human neutrophil responses. Garlic EO, allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC) directly activated Ca 2+ flux in neutrophils, with the most potent being AITC. Although 1,3-dithiane did not activate neutrophil Ca 2+ flux, this minor constituent of garlic EO stimulated neutrophil reactive oxygen species (ROS) production. In contrast, a close analog (1,4-dithiane) was unable to activate neutrophil ROS production. Although 1,3-dithiane-1-oxide also stimulated neutrophil ROS production, only traces of this oxidation product were generated after a 5 h treatment of HL60 cells with 1,3-dithiane. Evaluation of several phosphatidylinositol-3 kinase (PI3K) inhibitors with different subtype specificities (A-66, TGX 221, AS605240, and PI 3065) showed that the PI3K p110δ inhibitor PI 3065 was the most potent inhibitor of 1,3-dithiane-induced neutrophil ROS production. Furthermore, 1,3-dithiane enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase 3 α/β (GSK-3α/β), and cAMP response element binding (CREB) protein in differentiated neutrophil-like HL60 cells. Density functional theory (DFT) calculations confirmed the reactivity of 1,3-dithiane vs. 1,4-dithiane, based on the frontier molecular orbital analysis. Our results demonstrate that certain organosulfur compounds can activate neutrophil functional activity and may serve as biological response modifiers by augmenting phagocyte functions.

c-Jun N-terminal kinase (JNK) is activated by various brain insults and is implicated in neuronal injury triggered by reperfusion-induced oxidative stress. Some JNK inhibitors demonstrated neuroprotective potential in various models, including cerebral ischemia/reperfusion injury. The objective of the present work was to study the neuroprotective activity of a new specific JNK inhibitor, IQ-1S (11 H -indeno[1,2- b ]quinoxalin-11-one oxime sodium salt), in the model of global cerebral ischemia (GCI) in rats compared with citicoline (cytidine-5'-diphosphocholine), a drug approved for the treatment of acute ischemic stroke and to search for pleiotropic mechanisms of neuroprotective effects of IQ-1S. The experiments were performed in a rat model of ischemic stroke with three-vessel occlusion (model of 3VO) affecting the brachiocephalic artery, the left subclavian artery, and the left common carotid artery. After 7-min episode of GCI in rats, 25% of animals died, whereas survived animals had severe neurological deficit at days 1, 3, and 5 after GCI. At day 5 after GCI, we observing massive loss of pyramidal neurons in the hippocampal CA1 area, increase in lipid peroxidation products in the brain tissue, and decrease in local cerebral blood flow (LCBF) in the parietal cortex. Moreover, blood hyperviscosity syndrome and endothelial dysfunction were found after GCI. Administration of IQ-1S (intragastrically at a dose 50 mg/kg daily for 5 days) was associated with neuroprotective effect comparable with the effect of citicoline (intraperitoneal at a dose of 500 mg/kg, daily for 5 days).The neuroprotective effect was accompanied by a decrease in the number of animals with severe neurological deficit, an increase in the number of animals with moderate degree of neurological deficit compared with control GCI group, and an increase in the number of unaltered neurons in the hippocampal CA1 area along with a significant decrease in the number of neurons with irreversible morphological damage. In rats with IQ-1S administration, the LCBF was significantly higher (by 60%) compared with that in the GCI control. Treatment with IQ-1S also decreases blood viscosity and endothelial dysfunction. A concentration-dependent decrease (IC 50 = 0.8 ± 0.3 μM) of tone in isolated carotid arterial rings constricted with phenylephrine was observed after IQ-1S application in vitro. We also found that IQ-1S decreased the intensity of the lipid peroxidation in the brain tissue in rats with GCI. 2.2-Diphenyl-1-picrylhydrazyl scavenging for IQ-1S in acetonitrile and acetone exceeded the corresponding values for ionol, a known antioxidant. Overall, these results suggest that the neuroprotective properties of IQ-1S may be mediated by improvement of cerebral microcirculation due to the enhanced vasorelaxation, beneficial effects on blood viscosity, attenuation of the endothelial dysfunction, and antioxidant/antiradical IQ-1S activity.

A variety of natural compounds have been shown to modulate T cell receptor (TCR) activation, including natural sesquiterpene lactones (SLs). In the present studies, we evaluated the biological activity of 11 novel semi-synthetic SLs to determine their ability to modulate TCR activation. Of these compounds, α -epoxyarglabin, cytisinyl epoxyarglabin, 1 β ,10 α -epoxyargolide, and chloroacetate grosheimin inhibited anti-CD3-induced Ca 2+ mobilization and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in Jurkat T cells. We also found that the active SLs depleted intracellular glutathione (GSH) in Jurkat T cells, supporting their reactivity towards thiol groups. Because the zeta-chain associated tyrosine kinase 70 kDa (ZAP-70) is essential for TCR signaling and contains a tandem SH2 region that is highly enriched with multiple cysteines, we performed molecular docking of natural SLs and their semi-synthetic derivatives into the ZAP-70 binding site. The docking showed that the distance between the carbon atom of the exocyclic methylene group and the sulfur atom in Cys39 of the ZAP-70 tandem SH2 module was 3.04⁻5.3 Å for active compounds. Furthermore, the natural SLs and their derivatives could be differentiated by their ability to react with the Cys39 SH-group. We suggest that natural and/or semi-synthetic SLs with an α -methylene- γ -lactone moiety can specifically target GSH and the kinase site of ZAP-70 and inhibit the initial phases of TCR activation.

Essential oils (EOs) were obtained by hydrodistillation of various parts of Ferula ovina (Boiss.) Boiss., Ferula iliensis Krasn. ex. Korovin, and Ferula akitschkensis B. Fedtsch. ex Koso-Pol., collected in the flowering/budding and fruiting stages. Eight samples of EOs isolated from F. ovina and four samples from F. akitsckensis were analyzed by gas chromatography⁻mass spectrometry (GC-MS). The major constituents of F. ovina EOs were α-pinene (6.9⁻47.8%), β-pinene (1.5⁻7.1%), sabinene (0.1⁻20.5%), β-phellandrene (0⁻6.5%), trans -verbenol (0.9⁻7.4%), eremophilene (3.1⁻12%), and 6 Z -2,5,5,10-tetramethyl-undeca-2,6,9-trien-8-one (0⁻13.7%). The major constituents of F. akitsckensis EOs were α-pinene (0⁻46.2%), β-pinene (0⁻47.9%), sabinene (0⁻28.3%), eremophilene (0⁻10.6), β-caryophyllene (0⁻7.5%), himachalen-7-ol (0⁻28.2%), and an himachalol derivative (0⁻8.3%). Samples of EOs from F. ovina , F. iliensis , and F. akitsckensis were evaluated for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) pulse-field gel electrophoresis type USA300 (LAC). EOs from F. ovina exhibited the highest antibacterial activity compared to samples from other Ferula spp., with the most potent EOs being isolated from roots at the flowering and fruiting stages and stems at the fruiting stage (IC 50 values of 19.1, 20.9, and 22.9 µg/mL, respectively). Although EOs demonstrated concentration-dependent inhibition of MRSA growth, analysis of the major constituents (α-pinene, β-pinene, and sabinene) showed that they had low activity, suggesting that other components were likely responsible for the observed bioactivity of the unfractionated EOs. Indeed, correlation of the GC-MS data with antibacterial activity suggested that the putative components responsible for antibacterial activity were, either individually or in combination, eremophilene and trans -verbenol. Overall, these results suggest that the EOs from F. ovina could have potential for use as alternative remedies for the treatment of infectious diseases caused by MRSA.

Although humic acids (HAs) from peat exhibit various therapeutic properties, there is little information available concerning their physicochemical and antioxidant properties. To address this issue, nine different types of peat, including oligotrophic, mesotrophic, and minerotrophic peat samples, were used for isolation of HA fractions by basic (HAb) and pyrophosphate (HAp) extractions. Physical parameters of the HAs were analyzed by UV-Vis, fluorescent, infrared (IR), and electron paramagnetic resonance (EPR) spectroscopy. Average M r of the fractions ranged from 17.2 to 39.7 kDa, while their humification index (HIX) varied from 0.49 to 1.21. HAp fractions had a higher content of aromatic structures compared to HAb fractions. Moreover, HAp fractions had a significantly higher content of phenolic OH groups (3.6 ± 0.5 mmol/g) versus HAb (3.1 ± 0.5 mmol/g). All HA fractions exhibited antioxidant activity in radical scavenging and electrochemical assays, and their EPR signal had a single line with g = 2.0035, which is consistent with semiquinone type radicals. Furthermore, the HIX was found to be important in determining the number of semiquinone-type free radicals in the HA structures. Overall, these data provide a molecular basis to explain at least part of the beneficial therapeutic properties of peat-derived HAs., Competing Interests: The authors declare no conflict of interest.