Lipoxygenase Inhibitor Research Articles

Anti-inflammatory and Antimicrobial Potential of Three Natural Polyketides Isolated from Endophytic Fungus Phomopsis sp CAM212 against to Dysenteric Causing Pathogens

Aims: The present work aimed to evaluate the anti-amoebic, antibacterial, and anti-inflammatory potential of three natural polyketides from Phomopsis sp. CAM212.
 Study Design: Clinical isolates of E.histolytica, E.coli ATCC25922 strain, primary peritoneal mouse macrophages and three polyketides were used.
 Places and Duration of Study: Laboratory of Pharmacology and Toxicology, Laboratory of Medical Microbiology, Faculty of Science, University of Yaounde 1 between May and December 2022.
 Methodology: During this work, we evaluated the ability of three natural polyketides from Phomopsis sp to inhibit the growth of germs responsible for amoebic and bacillary dysentery. First, the anti-amoebic activity was carried out on clinical isolates of E. histolytica in polyxenic culture. Subsequently, we evaluated the antibacterial potential on a strain of E. coli ATCC25922. Finally, the anti-inflammatory potentials were evaluated on a primary culture of SC activated macrophages through inhibition of nitric oxide (NO) production, activation of phosphatase alcaline (ALP) and inhibition of 5-lipoxygenase (5-LOX).
 Results: It emerges from this work that among compounds, phomopsinin B, presented the highest anti-amoebic potential (84.4 % inhibition after 72h) and the highest antibacterial potential (MIC=12.5µg/mL and MBC/MIC=2). Phomopsini A and phomopsini A acetate showed moderate anti amoebic and antibacterial potentials. However, all these activities remain lower than that of metronidazole and ciprofloxacin (90% of amoebic inhibition after 72h; MIC=0.72µg/mL and MBC/MIC=4). Subsequently, all tested compounds were nontoxic on primary macrophages. Phomopsinin B exhibited a great anti-inflammatory potential through the inhibition of NO production (IC50=1.72±0.91µg/mL); inihibition of 5-LOX activity (IC50=36.97±7.12µg/mL) and activation of ALP activity (IC50=0.13±0.01µg/mL) as compared to Baicalin the standard. The anti-inflammatory potential of phomopsinin A and phomopsinin A acetate were lower compared to baicalin.
 Conclusion: Ultimately, among compounds tested, phomopsinin B exhibited the best anti-amoebic, antibacterial and ant-inflammatory potential similar to the respective standards within the limits of the tests carried out.

GC-MS Bioactive Compound Identification, in vitro Nutraceutical and Pharmacological Potential of Underutilized Leafy Vegetable (Ipomoea batatas leaf)

The search for dietary sources with potent biological activities has increasingly attracted considerable attention. Sweet potato leaves (SPL) are considered to be a leafy vegetable consumed by humans, which is not currently widely used, despite it possession drought tolerance, and the ability to grow in different climates and farming systems. This study evaluated the in vitro Nutraceutical and Pharmacological potential of underutilized leafy vegetable (Ipomoea batatas leaf). The phytochemicals, in vitro antioxidant and anti-inflammatory activities; mineral and vitamins compositions of the leaf were carried out using standard method. The bioactive compounds in Ipomoea batatas were identified using GC-MS. The results of the GC-MS profiling revealed n-Hexadecanoic acid (23.43%), Cyclotrisiloxane, hexamethyl (17.73%), and 16-Pregnenolone (11.30%) as the most abundant bioactive compounds in Ipomoea batata leaves. The mineral analysis showed that Ipomoea batata leaves contains favorable amount of macro elements such as Ca (915.40±9.50), K (2083.30±15.00), P (511.26±4.70), Mg (271.25±7.20), Na (9.98±1.06) and considerable amount of trace elements such as Fe (10.60±1.30), Mn (2.55±0.27), Zinc (2.70±0.11), and Cu (1.32±0.08) in which all were measured in miligram per 100g (mg/100). The vitamins such as of Vitamin A, vitamins C, vitamin E, niacin (B3), thiamine (B1), riboflavin (B2), pyridoxine (B6), vitamin D and vitamin K are present in trace amount in the leaves. The in vitro antioxidant activities of the extract showed a significant (p<0.05) dose dependent free radical scavenging activities. The anti-inflammatory analysis showed that the percent inhibition of hemolysis (20–100 µg/mL dry weight basis) was within the range of 4.1% to 18%. Percent inhibition of protein denaturation was within the range of 29.0–71.0%, Proteinase inhibitory activity was within the range of 16–30.0% and the lipoxygenase inhibition was within the range of 8.2–34.5%. This study provide validation for the usage of Ipomoea batatas leaf as new leafy vegetable with appreciable nutritional values and also provided credence to the bioactive compounds in leaf as potential novel drug candidate for pharmaceutical industries.

Novel Pyrimidine Derivatives as Antioxidant and Anticancer Agents: Design, Synthesis and Molecular Modeling Studies.

The heterocyclic ring system of pyrido [2,3-d]pyrimidines is a privileged scaffold in medicinal chemistry, possessing several biological activities. The synthesis of the pyrimidine derivatives was performed via the condensation of a suitable α,β-unsaturated ketone with 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate in glacial acetic acid. Chalcones were synthesized, as starting materials, via the Claisen-Schmidt condensation of an appropriately substituted ketone and an appropriately substituted aldehyde in the presence of aqueous KOH 40% w/v in ethanol. All the synthesized compounds were characterized using IR, 1H-NMR, 13C-NMR, LC-MS and elemental analysis. The synthesized compounds were evaluated for their antioxidant (DPPH assay), anti-lipid peroxidation (AAPH), anti-LOX activities and ability to interact with glutathione. The compounds do not interact significantly with DPPH but strongly inhibit lipid peroxidation. Pyrimidine derivatives 2a (IC50 = 42 μΜ), 2f (IC50 = 47.5 μΜ) and chalcone 1g (IC50 = 17 μM) were the most potent lipoxygenase inhibitors. All the tested compounds were found to interact with glutathione, apart from 1h. Cell viability and cytotoxicity assays were performed with the HaCaT and A549 cell lines, respectively. In the MTT assay towards the HaCaT cell line, none of the compounds presented viability at 100 μM. On the contrary, in the MTT assay towards the A549 cell line, the tested compounds showed strong cytotoxicity at 100 μM, with derivative 2d presenting the strongest cytotoxic effects at the concentration of 50 μΜ.

Effects of inhibition of 5-lipoxygenase and 12-lipoxygenase pathways on skeletal muscle fiber regeneration

We investigated the effect of inhibition of 5-lipoxigenase (LOX) and 12-LOX pathways on the regeneration of skeletal muscle fibers after injury induced by a myotoxin (MTX) phospholipase A2 from snake venom in an in vivo experimental model. Gastrocnemius muscles of mice injected with MTX presented an increase in 5-LOX protein expression, while 12-LOX was found to be a constitutive protein of skeletal muscle. Animals that received oral treatments with 5-LOX inhibitor MK886 or 12-LOX inhibitor baicalein 30 min and 48 h after MTX-induced muscle injury showed a reduction in the inflammatory process characterized by a significant decrease of cell influx and injured fibers in the degenerative phase (6 and 24 h after injury). In the beginning of the regeneration process (3 days), mice that received MK886 showed fewer new basophilic fibers, suggesting fewer proliferative events and myogenic cell fusion. Furthermore, in the progression of tissue regeneration (14–21 days), the mice treated with 5-LOX inhibitor presented a lower quantity of central nucleus fibers and small-caliber fibers, culminating in a muscle that is more resistant to the stimulus of fatigue during muscle regeneration with a predominance of slow fibers. In contrast, animals early treated with the 12-LOX inhibitor presented functional fibers with higher diameters, less resistant to fatigue and predominance of fast heavy-chain myosin fibers as observed in control animals. These effects were accompanied by an earlier expression of myogenic factor MyoD. Our results suggest that both 5-LOX and 12-LOX pathways represent potential therapeutic targets for muscle regeneration. It appears that inhibition of the 5-LOX pathway represses only the degenerative process by reducing tissue inflammation levels. Meanwhile, inhibition of the 12-LOX pathway also favors the anticipation of maturation and earlier recovery of muscle fiber activity function after injury.

Future Perspectives of Oxytosis/Ferroptosis Research in Neurodegeneration Diseases.

The current report briefly summarizes the existing hypotheses and relevant evidence of oxytosis/ferroptosis-mediated cell death and outlines future perspectives of neurodegeneration research. Furthermore, it highlights the potential application of specific markers (e.g., activators, inhibitors, redox modulators, antioxidants, iron chelators) in the study of regulatory mechanisms of oxytosis/ferroptosis. It appears that these markers may be a suitable option for experimental investigations targeting key pathways of oxytosis/ferroptosis, such as the inhibition of the cystine/glutamate antiporter/glutathione/glutathione peroxidase 4 axis, glutamate oxidative toxicity, glutathione depletion, iron dyshomeostasis, iron-mediated lipid peroxidation, and others. From a clinical perspective, an innovative research approach to investigate the oxytosis/ferroptosis pathways in cells of the central nervous system and their relationship to neurodegenerative diseases is desirable. It is necessary to expand the existing knowledge about the molecular mechanisms of neurodegenerative diseases and to provide innovative diagnostic procedures to prevent their progression, as well as to develop effective neuroprotective treatment. The importance of preclinical studies focused predominantly on oxytosis/ferroptosis inhibitors (iron chelators or lipoxygenase inhibitors and lipophilic antioxidants) that could chelate iron or inhibit lipid peroxidation is also discussed. Specifically, this targeted inhibition of neuronal death could represent a potential therapeutic strategy for some neurodegenerative diseases.

Gochnatia glutinosa (D.Don) D.Don ex Hook. & Arn.: A plant with medicinal value against inflammatory disorders and infections

Gochnatia glutinosa is a shrub that grown in the Argentinean semiarid region (Monte region) used in the ancestral medicine as an antiseptic and anti-inflammatory agent.This study was aimed to examine the morpho-anatomical characteristics of G. glutinosa aerial parts, identify the chemical composition of traditionally used preparations to assess its pharmacobotanical characterization and evaluate its activity as antiseptic and anti-inflammatory to give scientific support to its traditional uses. G. glutinosa morpho-anatomical description was performed following standard histological techniques. Tincture and infusion of its aerial parts were prepared and were subjected to phytochemical analysis. Xanthine oxidase (XOD) and lipoxygenase (LOX) inhibition experiments, as well as ABTS•+, superoxide radical, and hydrogen peroxide scavenging activity, were carried out. The growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA) strains was also determined.The morpho-anatomical traits of G. glutinosa leaves and stems were reported for the first time. The medicinal preparations exhibited a large amount of phenolic chemicals mainly flavonoids such as rhamnetin, arcapillin, rhamnacin, hesperetin, isorhamnetin, centaureidin, europetin 7-O-mehylmyricetin, cirsiliol, sakuranetin, genkwanin and eupatorine and also phenolic acids and diterpenoid derivatives. Both preparations had free radical scavenging activity and were able to reduce both XOD and LOX activity, indicating their anti-inflammatory properties. Besides, tincture was effective against all MRSA strains (MIC values ranging from 60 to 240 g DW/mL).The results obtained in this work scientifically support the medicinal popular use of G. glutinosa as an antiseptic and anti-inflammatory. The identification of bioactive compounds and their morpho-anatomical description contribute to the quality control of this medicinal plant from Argentine Calchaquí Valley.

Effective Synthesis and Biological Evaluation of Dicoumarols: Preparation, Characterization, and Docking Studies.

A series of 3,3-arylidene bis (4-hydroxycoumarins) 2 were synthesized by the reaction of aromatic aldehydes with 4-hydroxycoumarin using dodecylbenzenesulfonic acid as Brønsted acid-surfactant catalyst in aqueous media and under microwave irradiation. The present method is operationally simple and the use of water as the reaction medium makes the process environmentally benign. The epoxydicoumarins 5 were then obtained with a good yield by heating 3,3'-arylidenebis-4-hydroxycoumarins 2 in acetic anhydride. Techniques such as elemental analysis, 1H, 13C-1H NMR, and infrared spectroscopy were employed to characterize these compounds. The synthesized compounds displayed good antibacterial potential against Escherichia coli (ATCC 25988), Pseudomonas aeruginosa (ATCC 27853), Klebsilla pneumonia (ATCC 700603), Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (ATCC 43300) and Candida albicans (ATCC 14053). The MIC values of 23 mg/mL for compound 5e against Escherichia coli (ATCC 25988) and 17 mg/mL for 2a were observed. Furthemore, a molecular docking simulation has been performed to evaluate the antibacterial activities and the probable binding modes of the studied compounds 2a-f and 5a-g toward the active sites of a series of well known antibacterial targets. Among the investigated compounds, the binding modes and docking scores demonstrate that 2a has the most antibacterial and antifungal activities. Additionally, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS has been tested for their ability to scavenge hydrogen peroxide and free radicals. According to our results, these compounds exhibit excellent radical scavenging properties. Furthermore, compounds 2-5 were evaluated for anti-inflammatory activity by indirect haemolytic and lipoxygenase inhibition assays and revealed good activity.

Characterization, Antioxidant Activity, and In Silico Molecular Docking of Chitosan from Snail Shell Waste by Ultrasonic Technique

Snails are often found in Indonesia, especially at Kediri, but the snail shell has no commercial value. This research report describes the characterization and antioxidant activity of chitosan from snail shell waste (chitosan-SSW) by ultrasonic technique and analyzes the potential of chitosan as an inhibitor of receptors of free radicals using an in silico molecular docking method. Characterization of chitosan-SSW was performed to analyze the content of water, protein, and functional groups as well as molecular weight, particle size, mor-phology, antioxidant activity, and in silico molecular docking. We found that chitosan-SSW with ultrasonic treatment had a high degree of deacetylation (DD) and high molecular weight (MW). The characteristic of chitosan-SSW was found to be as follows: water content of 0.43 %, protein content of 1.59 %, molecular weight of 2.198 kDa, and deacetylation degree value of 79.50 %. Importantly, chitosan-SSW had high antioxidant activity to potentially reduce free radical of DPPH with IC50 value of 2.44 µg/mL. Chitosan is predicted to have the potential as an inhibitor of lipoxygenase, CYP2C9, and NADPH-oxidase.

Thiocarbohydrazone and Chalcone-Derived 3,4-Dihydropyrimidinethione as Lipid Peroxidation and Soybean Lipoxygenase Inhibitors.

The potential of the 4,6-diphenyl-3,4-dihydropyrimidine-2(1H)-thione (abbreviated as KKII5) and (E)-N'-benzylidenehydrazinecarbothiohydrazide (abbreviated as DKI5) compounds as possible drug leads is investigated. KKII5 and DKI5 are synthesized in high yield of up to 97%. Their structure, binding in the active site of the LOX-1 enzyme, and their toxicity are studied via joint experimental and computational methodologies. Specifically, the structure assignment and conformational analysis were achieved by applying homonuclear and heteronuclear 2D nuclear magnetic resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and density functional theory (DFT). The obtained DFT lowest energy conformers were in agreement with the NOE correlations observed in the 2D-NOESY spectra. Additionally, docking and molecular dynamics simulations were performed to discover their ability to bind and remain stabile in the active site of the LOX-1 enzyme. These in silico experiments and DFT calculations indicated favorable binding for the enzyme under study. The strongest binding energy, -9.60 kcal/mol, was observed for dihydropyrimidinethione KKII5 in the active site of LOX-1. ADMET calculations showed that the two molecules lack major toxicities and could serve as possible drug leads. The redox potential of the active center of LOX-1 with the binding molecules was calculated via DFT methodology. The results showed a significantly smaller energy attachment of 2.8 eV with KKII5 binding in comparison to DKI5. Thus, KKII5 enhanced the ability of the active center to receive electrons compared to DKI5. This is related to the stronger binding interaction of KKII5 relative to that of DK15 to LOX-1. The two very potent LOX-1 inhibitors exerted IC50 19 μΜ (KKII5) and 22.5 μΜ (DKI5). Furthermore, they both strongly inhibit lipid peroxidation, namely, 98% for KKII5 and 94% for DKI5.

Extraction of Phytochemical Constituents from the Leaves of Vitex trifolia in the Inhibition of Lipoxygenase, Cyclooxygenase, Tyrosinase and Xanthine Oxidase

Background: Herbal medicines are often used to stimulate the immune system in an attempt to prevent disease, as well as to induce specific cures. The use of phyto medicines is becoming more scientifically based, with increasing emphasis placed on proven product safety and efficacy. Methods: In the present study, the leaves of Vitex trifolia were extracted with distilled water, 1M Na2CO3 and 70% methanol. The extracted contents were screened to identify the presence of various phytochemicals. The contents of Na2CO3 and methanol were filtered, centrifuged and the supernatant was collected. The supernatant of Na2CO3 extract was collected and the alkaloid layer was separated in a separating funnel using alcohol. The contents were analysed for thin layer chromatography and phytochemical constituents. Result: Thin layer chromatography was performed at360 nm the Rf value were 0.953, 0.230, 0.184, 0.153, 0.123 and in 240 nm Rf values 0.953, 0.338, 0.184, 0.153, 0.123 were obtained. The methanol extract was condensed at 40oC and dissolved in chloroform. Rf at 360 nm of 0.714, 0.396, 0.158 and at 240 nm with Rf values 0.714, 0.492, 0.349. The activity of extracts on Xanthine oxidase, tyrosinase and cyclooxygenase were estimated and the inhibition percentage of all the three was calculated as 59.34, 61.23, 71.02 and 51 respectively. The values obtained with flavanoid extracts for the same set of enzymes were 70.22, 81.48, 94.8 and 74.36 respectively. In this study, inhibition properties of flavanoid extracts were greater than that of alkaloid extracts.

In vitro Evaluation of Antioxidant and Antibacterial Activities of Eco-friendly Synthesized Silver Nanoparticles using Quercus robur Bark Extract.

This study reports a simple, cost-effective, and environmentally friendly method to obtain silver nanoparticles (AgNPs) using an aqueous extract of Quercus robur bark. AgNPs synthesis conditions such as silver nitrate concentration, extract:AgNO3 volume ratio, pH, temperature, and reaction time have been examined. After optimizing the synthesis, the obtained AgNPs were characterized by different methods such as UV-Vis, TEM, EDX, and FTIR. The antioxidant activity was evaluated using lipoxygenase inhibition capacity and inhibition of erythrocyte hemolysis mediated by peroxyl free radicals tests. The antimicrobial potential of the samples was tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The AgNPs synthesis process is influenced by reaction conditions, the optimum established values being, in this case: concentration of 3 mM AgNO3, 1:9 extract: AgNO3 volume ratio, pH value of 6, 60 ºC temperature, and 90 minutes stirring time. The shape of the synthesized AgNPs was predominantly spherical, with an average size of 50 nm. The SPR band at 432 nm, the strong EDX signal at ~ 3 keV and the zeta potential of -13.88 mV revealed the formation of AgNPs and electrostatic stabilization of the colloidal solution. FTIR analysis confirmed the participation of molecules from the extract in the synthesis and stabilization of AgNPs. The obtained nanoparticles showed improved antioxidant, antifungal and antibacterial activities compared to the extract. The results open the possibility of exploring new applications of nanoparticles obtained via green synthesis.

Prospects for Pharmaceutical of Zingiber officinale Extracts and Fractions by Analysis of the Chemical Markers, Safety Profile, Anti-Lipoxygenase, and Analgesic Activity

Ginger (Zingiber officinale) has gained recognition for its flavoring uses and its numerous health benefits. Ginger can be used in functional foods and nutraceuticals alone or combined with other herbal plants. This study aims to validate the pharmaceutical potential of the Zingiber officinale variety from the Philippines by examining the chemical composition, acute oral toxicity, anti lipoxygenase, and analgesic activities of its extracts and fractions. 6, 8, 10-Gingerols and 6, 8, 10-Shogaols were identified by high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in methanol and hexane extracts. Acute oral toxicity in mice indicated the median lethal dose (LD50) of methanol and hexane extracts of Zingiber officinale, and the 1% IPA-hexane and 50% IPA-hexane fractions obtained by solid phase extraction (SPE) was greater than 2000 mg/kg body weight. The 1% IPA-hexane fraction altered the behavioral pattern of the animals. The anti-inflammatory activity was analyzed by lipoxygenase (LOX) inhibition assay. The IC50 value for the inhibitory action of crude methanol extract on LOX was 218.00 µg/mL. The crude hexane extract IC50 value was 57.40 µg/mL. SPE fractionation of hexane extracts resulted in higher IC50 values; 72.14 µg/mL for the 1% IPAhexane fraction and 98.65 µg/mL for the 50% IPA-hexane fraction. Through in-vitro suppression of LOX, the hexane crude extracts showed more effective anti-inflammatory properties. The analgesic effect was measured using acetic acid-induced writhing in mice. All doses of the methanol and hexane extract inhibited writhing by a percentage ranging from 91-100%. The SPE fractions of the hexane extract have 78-99% inhibition. Both extracts and fractions demonstrated strong analgesic effects in the mice models.

General Approach to Identify, Assess, and Characterize Inhibitors of Lipid Peroxidation and Associated Cell Death.

Lipid peroxidation (LPO) is associated with a variety of pathologies and drives a form of regulated necrosis called ferroptosis. There is much interest in small-molecule inhibitors of LPO as potential leads for therapeutic development for neurodegeneration, stroke, and acute organ failure, but this has been hampered by the lack of a universal high-throughput assay that can identify and assess candidates. Herein, we describe the development and validation of such an approach. Phosphatidylcholine liposomes loaded with ∼10% phospholipid hydroperoxide and STY-BODIPY, a fluorescent signal carrier that co-autoxidizes with polyunsaturated phospholipids, are shown to autoxidize at convenient and constant rates when subjected to an optimized Fe2+-based initiation cocktail. The use of this initiation system enables the identification of each of the various classes of LPO inhibitors which have been shown to rescue from cell death in ferroptosis: radical-trapping antioxidants (RTAs), peroxidase mimics, and iron chelators. Furthermore, a limited dose-response profile of inhibitors enables the resolution of RTA and non-RTA inhibitors─thereby providing not only relative efficacy but mechanistic information in the same microplate-based experiment. Despite this versatility, the approach can still be used to estimate rate constants for the reaction of RTAs with chain-propagating peroxyl radicals, as demonstrated for a representative panel of RTAs. To illustrate the utility of this assay, we carried out a preliminary investigation of the 'off-target' activity of several ferroptosis suppressors that have been proposed to act independently of inhibition of LPO, including lipoxygenase inhibitors, cannabinoids, and necrostatins, the archetype inhibitors of necroptosis.

Kitul, a food plant with antidiabetic-like effects: Reduction of intracellular reactive species in glucose-stimulated RIN-5F pancreatic β-cells and mitigation of pro-inflammatory mediators in activated RAW 264.7 macrophages

Kitul (Caryota urens L.) inflorescences are broadly used for sweet sap production in Asian countries and Kitul food products are known as being suitable for diabetic patients. Considering the strong ability to inhibit α-glucosidase, we hypothesize that kitul antidiabetic properties might also involve the modulation of inflammatory pathways and hyperglycaemia-induced oxidative damage. Hence, the effects of an inflorescence’s methanol extract were investigated in glucose-stimulated pancreatic cells (RIN-5F) and LPS-stimulated RAW 264.7 macrophages. The extract reduced the overproduction of intracellular reactive species in pancreatic cells and also NO, L-citrulline and IL-6 levels in LPS-stimulated RAW 264.7 macrophages. Inhibition of 5-lipoxygenase (IC50 = 166.1 µg/mL) through an uncompetitive manner was also recorded upon treatment with C. urens inflorescences extract. The phenolic profile of the inflorescences was characterized by HPLC-DAD, six hydroxycinnamic acids being identified and quantified. Overall, our data provide additional evidence on the pleiotropic mechanisms of Kitul inflorescences as an antidiabetic agent.

Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

2,9-Dimethyl-4H-oxazolo[5’,4’:4,5]pyrano[3,2-f]quinolin-4-one

The new 2,9-dimethyl-4H-oxazolo[5’,4’:4,5]pyrano[3,2-f]quinolin-4-one was successfully prepared through the three-component iodine-catalyzed reaction of n-butyl vinyl ether with the new 8-amino-2-methyl-4H-chromeno[3,4-d]oxazol-4-one. The latter was prepared by the reduction of 2-methyl-8-nitro-4H-chromeno[3,4-d]oxazol-4-one with Pd/C in a hydrogen atmosphere. The above nitro compound was synthesized by the condensation of N-(4-hydroxy-6-nitro-2-oxo-2H-chromen-3-yl)acetamide with P2O5 under microwave irradiation. The above acetamide derivative was prepared during the nitration of 2-methyl-4H-chromeno[3,4-d]oxazol-4-one with H2SO4 and KNO3. The structure of the newly synthesized compounds was confirmed by FT-IR, LC-MS, 1H-NMR, and 13C-NMR analyses. Preliminary biological tests show significant anti-lipid peroxidation activity for the title compound and the other synthesized new intermediates, as well as interesting soybean lipoxygenase inhibition for acetamide 2 (IC50 55 μM) and nitro-compound 3 (IC50 27 μM).

Antiallergic Effects of Callerya atropurpurea Extract In Vitro and in an In Vivo Atopic Dermatitis Model.

(1) Background: Callerya atropurpurea is found in Laos, Thailand, and Vietnam. Although the anti-inflammatory action of C. atropurpurea has been investigated, the functions of this plant in allergic responses are not understood. Here, we explored the antiallergic mechanism of C. atropurpurea ethanol extract (Ca-EE) using in vitro assays and an in vivo atopic model. (2) Methods: The constituents of Ca-EE were analyzed using GC/MS. Inhibition of lipoxygenase and β-hexosaminidase activity was examined, and the expression of inflammatory genes was measured by quantitative real-time PCR. The regulatory roles of Ca-EE in IgE/FcεRI signaling were examined by Western blotting. The DNCB-induced atopic dermatitis mouse model was performed with histological analysis. (3) Results: Ca-EE comprised cis-raphasatin, lupeol, some sugars, and fatty acids. In RBL-2H3 cells, treatment with Ca-EE significantly reduced the activities of lipoxygenase and β-hexosaminidase, as well as cytokine gene expression. IgE-mediated signaling was downregulated by blocking Lyn kinases. Moreover, Ca-EE effectively inhibited allergic symptoms in the DNCB-induced atopic dermatitis model without toxicity. (4) Conclusions: Ca-EE displayed antiallergic activities through regulating IgE/Lyn signaling in RBL-2H3 cells and a contact dermatitis model. These results indicate that Ca-EE could be effective for allergic disease treatment.

New neuroprotective sesquiterpene lactate esters from carotol biotransformation

Biotransformation of carotol (1), the major sesquiterpenoid of carrot fruit essential oil, by Rhizopus oryzae yielded two new lactate derivatives: 9α-hydroxydaucol-9-lactate (2) and 9α-hydroxydaucol-3,9-dilactate (3). The structures of 1-3 were characterized by 1 & 2D NMR spectroscopy in conjunction with HR-ESIMS. Cholinergic deficiency and neuroinflammation are key detrimental processes involved in the etiology of Alzheimer's disease (AD). This study demonstrated that compounds 1-3 in addition to the essential oil showed considerable in-vitro inhibition of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and butyrylcholinesterase (BChE). Metabolite 2 showed improved inhibitory activities (9.03, 0.56, and 0.64 μM) against COX-2, 5-LOX, and BChE enzymes, compared to the parent compound, carotol 1 (82.57, 2.11, and 0.74 μM, respectively). The results were correlated to the in-silico binding interactions with the proteins of these enzymes by molecular docking. Carrot essential oil and carotol derivatives are suggested by this study as a potential treatment for neurodegenerative disease associated with neuroinflammation.

Exploring the Medicinal Potential of Achillea grandifolia in Greek Wild-Growing Populations: Characterization of Volatile Compounds, Anti-Inflammatory and Antioxidant Activities of Leaves and Inflorescences.

Various species of the genus Achillea L. (Asteraceae) are traditionally used worldwide for wound healing against diarrhea, flatulence, and abdominal pains, as diuretic and emmenagogue agents. In the present study, the essential oils (EOs) obtained separately from the leaves and inflorescences of wild-growing Achillea grandifolia Friv. from Mt. Menoikio and Mt. Pelion (Greece) were analyzed by Gas Chromatography-Mass Spectrometry. The major compounds found in EOs of A. grandifolia inflorescences from Mt. Menoikio were as follows: cis-thujone (36.9%), 1,8-cineole (11.9%), camphor (10.0%), ascaridole (7.3%), α-terpinene (6.4%), sabinene (4.1%), trans-thujone (3.6%), and cis-jasmone (3.4%). In leaves from Mt. Menoikio, they were as follows: cis-thujone (50.8%), 1,8-cineole (20.0%), trans-thujone (5.5%), camphor (5.5%), borneol (3.6%), and α-terpineol (3.1%). In inflorescences from Mt. Pelion, they were as follows: camphor (70.5%), camphene (5.9%), cis-jasmone (3.2%), bornyl acetate (3.2%). In leaves from Mt. Pelion, they were as follows: camphor (83.2%), camphene (3.9%), and borneol (3.7%). Subsequently, the samples were first time tested for their antioxidant activities with the interaction of EOs with DPPH (2,2-diphenyl-1-picrylhydrazyl) and their inhibition of lipid peroxidation, as well as for their anti-inflammatory activity through the soybean LOX (lipoxygenase) inhibition. All of the examined samples were found effective. A. grandifolia leaves presented the highest antioxidant potential according to the DPPH method, and the highest percentage of LOX inhibition. The study herein investigated for the first time the leaves and the inflorescences of A. grandifolia separately, and the results generally align with similar studies from neighboring countries (Turkey and Serbia) in terms of the yields and categorization of main EO compounds (oxygenated monoterpenes). However, the findings were not in agreement with previously studied Greek material, as a higher amount of cis-thujone and lower antioxidant activity are reported herein. Both the EOs of inflorescences and the leaves of the wild-growing population collected from Mt. Menoikio were characterized by a high quantity of cis-thujone (36.9% and 50.8%, respectively).

Phytochemical profile and anti-inflammatory activity of a commercially available Rhodiola rosea root extract

Rhodiola rosea roots and rhizomes hold an important place in the folk medicines of Russia, Scandinavia, Mongolia, and China as a health supplement for stimulating the nervous system, enhancing physical and mental performances, and nowadays they constitute the active ingredient in many popular commercial preparations sold worldwide as food additives, pharmaceutical remedies, and drinks. This study was aimed at providing a detailed phytochemical characterization of the Rhodiola 5%, a commercially available extract of R. rosea roots, and resulted in the characterization of 18 secondary metabolites, including 13 polyphenols and 6 terpenoids, and in the discovery of the new rhodiosidin (5), the first R. rosea metabolite to show both terpenoid and cinnamoyl moieties. The 5-lipoxygenase inhibiting activity of the main components was characterized and disclosed that rosiridin (6), kenposide A and rosavins are mainly responsible for this activity of the extract.