5-LOX Enzyme Research Articles

The Antimicrobial, Antioxidant and Anti-Inflammatory Evaluation of Marrubium cuneatum Banks & Sol. Extracts & Fractions

Objective: This study aimed to determine the antimicrobial, antioxidant, and anti-inflammatory potential of the Marrubium cuneatum aqueous and extracts, in addition to the fractions acquired from the liquid-liquid fractionation, respectively. Method: The plant extracts were obtained using maceration, which were separated into their fractions based on polarity using the liquid-liquid fractionation method. The in vitro antimicrobial activity was evaluated using the microdilution method. The antioxidant activity was assessed using DPPH•, ABTS+, and CUPRAC assays. The anti-inflammatory activity of the extracts and fractions were evaluated by examining their effects on lipoxygenase (5-LOX) enzyme inhibition. Results: In the antioxidant assays, the n-butanol fraction of the methanolic extract and the ethyl acetate fraction of the aqueous extract showed activity in all tests, followed by the ethyl acetate fraction of the methanolic extract, respectively. In the antimicrobial assays, no remarkable inhibitions were observed compared to the reference compounds. It was noticed that the extracts and fractions showed no effect on the inhibition of the lipoxygenase enzyme. Conclusion: The ethyl acetate and n-butanol fractions showed relatively strong antioxidant effects, however, no significant antimicrobial activity was observed. In terms of anti-inflammatory activity, it was determined that the plant did not exhibit any anti-inflammatory effect through the lipoxygenase enzyme suggesting further detailed research on other fractions or purified compounds of the plant material.

Synthesis of Anti-Inflammatory Drugs' Chalcone Derivatives and a Study of Their Conformational Properties Through a Combination of Nuclear Magnetic Resonance Spectroscopy and Molecular Modeling.

In this study, two chalcone analogs were synthesized through in silico and experimental methods, and their potential to inhibit the lipoxygenase enzyme, which plays a role in the inflammation pathway, was assessed. Specifically, this study is a continuation of previous research in which chalcone derivatives were synthesized and characterized. In the current work, we present the re-synthesis of two chalcones, with a focus on their docking studies, NMR analysis, and dynamic simulations. The structure of each chalcone was elucidated through a combination of Nuclear Magnetic Resonance (NMR) and Density Functional Theory (DFT). The substituent effect on the absorption spectrum of the two chalcone derivatives was studied. A "LOX-chalcone" complex, predicted by docking studies, was further examined using molecular dynamics (MD) simulations to evaluate the stability of the complex. After fully characterizing the "LOX-chalcone" complexes in silico, the atomic details of each chalcone's interaction with LOX-1 and 5-LOX were revealed through Saturation Transfer Difference (STD) NMR (Nuclear Magnetic Resonance). Finally, their selectivity profile was investigated against human 15-LOX-1 and general Lipoxidase activity. The in silico methods suggest that chalcones could be promising lead compounds for drug designs targeting the LOX enzyme.

Systematic insight into the dual COX-2/5-LOX inhibitory mechanism of Duhuo Jisheng decoction for treatment of osteoarthritis based on in silico and bioassay.

Traditional Chinese medicine (TCM) is frequently used to treat osteoarthritis (OA). Duhuo Jisheng decoction (DHJSD), a Chinese patent medicine, was commonly used Chinese herbal formula for the treatment of OA. In Western medicine, dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme has been proved to be a promising strategy to treat inflammatory diseases with reduced side effects. To elucidate the dual action mechanism of DHJSD targeting COX-2 and 5-LOX against OA. DHJSD, containing 1495 compounds was screened using a virtual screening approach based on molecular docking. The inhibitory effect of hit compounds against COX-2 and 5-LOX was validated using enzyme-based assays. In vitro, rat chondrocytes were treated with IL-1β (10ng/mL) for 24h to induce OA model in vitro. The chondrocyte viability was evaluated using an CCK-8 assay. ELISA was used to detect inflammatory factors expression. Immunofluorescence was used to assess the expression level of collagen II and MMP-13. In addition, a rat cartilage explants culture model was established, and safranin O and HE staining analysis were carried to assess cartilage matrix degradation and cartilage damage, respectively. In vivo, carrageenan-induced paw edema assay was used to examine anti-inflammatory activity, and the gastric ulcerogenic effect was further detected. Finally, Molecular dynamics simulations and binding free energy analysis were carried to explore the binding mechanism. 13 compounds from DHJSD were identified as promising candidates by a virtual screening approach. Among these candidates, three hits 7,4'-dimethoxyisoflavone, genistein, and fraxetin displayed dual inhibition of COX-2 and 5-LOX. Further in vitro assay indicated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin could inhibit PGE2, LTB4, TNF-α, IL-6, or NO production in IL-1β-induced chondrocytes. In addition, the three compounds reduced IL-1β-induced degradation of collagen II and expression of MMP-13 in rat chondrocytes. The results of anti-inflammatory activity of the three compounds in vivo showed that the highest anti-inflammatory activity with edema inhibition percentages of 50.00%, 56.00%, and 51.00% after 3h, respectively. Moreover, it was found that 7,4'-dimethoxyisoflavone, genistein, and fraxetin have a superior gastric safety profile comparable to indomethacin. Finally, molecular dynamics simulations, binding free energy analysis, and detailed interaction mode demonstrated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin interacted well with both COX-2 and 5-LOX. 7,4'-dimethoxyisoflavone, genistein, and fraxetin from DHJSD with excellent anti-inflammatory effects and no gastric ulceration effects, which helps to explain the dual action mechanism and potential material basis of DHJSD in treating OA and provide evidence to support DHJSD's clinical use.

Dual anti-inflammatory activities of COX-2/5-LOX driven by kratom alkaloid extracts in lipopolysaccharide-induced RAW 264.7 cells

Cyclooxygenase (COX) and lipoxygenase (LOX) enzymes play a pivotal role in producing pro-inflammatory eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs), in the inflammation process. Mitragynine is a primary alkaloid contained in the kratom’s leaves and has been reported to show anti-inflammatory activity by suppressing COX-2 mRNA translation to lowering PGs synthesis. In this study, the Kratom’s alkaloid extract containing ~ 46% mitragynine was found to exhibit dual inhibition activity towards COX-2/5-LOX enzymes at concentrations below 25 ppm in the LPS-induced RAW 264.7 macrophage cells. At these levels, no cell toxicity was observed while the cells became death (e.g., 10–46% viability at 50–100 ppm) and only COX-2 inhibition activity was observed after exposed with more than 25 ppm of alkaloid extract. In contrast, the methanolic-crude extract of Kratom’s leaf containing ~ 5% mitragynine showed no inhibition toward COX-2/5-LOX enzymes and did not toxic onto the cells, even after treated at 100 ppm. The alkaloid extract suppressed several antiinflammation parameters, including ROS (64% reduction at 25 ppm), NO (30% reduction at 25 ppm), TNF-α (~ 50% reduction at 25 ppm), and IL-6 production (60% reduction at 6.25 ppm). In silico molecular studies indicated strong binding affinity of Kratom alkaloids to COX-2 and 5-LOX active sites, supporting the Kratom’s alkaloids to have great potential dual inhibition activity towards COX-2/5-LOX enzymes and to be developed as a safer NSAIDs with fewer side effects.

Synthesis and biological evaluation of diclofenac acid derivatives as potential lipoxygenase and α-glucosidase inhibitors.

Inflammation is a complex physiological response associated with the onset and progression of various disorders, including diabetes. In this study, we synthesized a series of diclofenac acid derivatives and evaluated their potential anti-diabetic and anti-inflammatory activities. The compounds were specifically assessed for their ability to inhibit 15-lipoxygenase (15-LOX) and α-glucosidase enzymes. The structures of synthesized derivatives were confirmed through 1H nuclear magnetic resonance (NMR), 13C-NMR and high-resolution mass spectrometry (electron ionization) analysis. All these synthesized derivatives exhibited varying degrees of inhibitory activity against LOX, when compared with standard drugs, compounds 5a (half-maximal inhibitory concentration (IC50) 14 ± 1 µM), 5b (IC50 61 ± 1 µM) and 7c (IC50 67 ± 1 µM) showed good activity against the LOX enzyme. While the α-glucosidase inhibitory results revealed that most of the compounds exhibited significant activity when compared with the standard drug acarbose (376 ± 1 µM). The most potent compounds as α-glucosidase inhibitors were 7b (3 ± 1 µM), 4b (5 ± 1 µM), 7a (7 ± 1 µM) and 8b (11 ± 1 µM). All these active compounds were found to be least toxic and maintained the mononuclear cells viability at 96-97% compared with that of controls as determined by multi-transaction translator assay. Molecular docking studies further reiterated the significance of these 'lead' compounds with great potential against the target enzymes in the process of drug discovery.

Role of Callistemon citrinus Leaf Phytosomes Against Oxidative Stress and Inflammation in Rats Fed with a High-Fat-Fructose Diet.

Phytosomes are used as vehicles that carry plant extracts. They exhibit biological activities and possess better bioavailability, bioabsorption, and lower toxicity than drugs. Obesity is an inflammatory state in which oxidative stress is present, which triggers severe effects on the body's organs. This study aimed to evaluate the impact of the extract and phytosomes of Callistemon citrinus on oxidative stress and inflammation in the liver and heart of Wistar rats fed with a high-fat-fructose diet. Phytosomes containing the extract of leaves of C. citrinus were prepared. The antioxidant, pro-inflammatory enzymes, and biomarkers of oxidative stress were evaluated. Among the groups, only the high-fat-fructose group presented an increase in the COX-2, 5-LOX, and MPO inflammatory enzymes, while the XO enzyme exhibited decreased activity. The groups were fed a hypercaloric diet for 15 weeks while orlistat, C. citrinus extract, and phytosomes were administered at three different concentrations, exhibiting enzyme activities similar to those of the control group. It was also observed that the lowest concentration of phytosomes had a comparable effect to the other concentrations. Callistemon citrinus extract can modulate the activities of enzymes involved in the inflammation process. Furthermore, small doses of phytosomes can serve as anti-inflammatory agents.

Activation of pro-resolving pathways mediate the therapeutic effects of thymosin beta-4 during Pseudomonas aeruginosa-induced keratitis.

Current treatments for bacterial keratitis fail to address the sight-threatening inflammatory host response. Our recent work elucidating the therapeutic mechanisms of adjunctive thymosin beta-4 (Tβ4) in resolving inflammation and infection in bacterial keratitis revealed modulation of effector cell function and enhanced bacterial killing. The current study builds upon the observed effects on effector cell function by investigating the impact of Tβ4 on specialized pro-resolving lipid mediator (SPM) pathways as they play a significant role in inflammation resolution. Using a well-established in vivo model of Pseudomonas aeruginosa-induced bacterial keratitis, we assessed key enzymes (5-LOX and 12/15-LOX) involved in SPM pathway activation, SPM end products (lipoxins, resolvins), and receptor levels for these mediators. In vitro validation using LPS-stimulated murine monocyte/MΦ-like RAW 264.7 cells and siRNA to inhibit Tβ4 and LOX enzymes was carried out to complement our in vivo findings. Findings from our in vivo and in vitro investigations demonstrated that adjunctive Tβ4 treatment significantly influences enzymes and receptors involved in SPM pathways. Further, Tβ4 alone enhances the generation of SPM end products in the cornea. Our in vitro assessments confirmed that Tβ4-enhanced phagocytosis is directly mediated by SPM pathway activation. Whereas Tβ4-enhanced efferocytosis appeared to be indirect. Collectively, these findings suggest that the therapeutic effect of Tβ4 resolves inflammation through the activation of SPM pathways, thereby enhancing host defense and tissue repair. Our research contributes to understanding the potential mechanisms behind Tβ4 immunoregulatory function, pointing to its promising ability as a comprehensive adjunctive treatment for bacterial keratitis.

Dual COX/ 5-LOX inhibitory potential and antioxidant activities by extracts isolated from Melia Azedarach L. leaves

This study was aimed at evaluating the dual cyclooxygenase (COX) and 5-Lipoxygenase (5-LOX) enzyme inhibition and antioxidant activity of the crude extract (CE) and various solvent fractions from Melia azedarach leaves. Multiple solvents were used to fractionate the 80% ethanolic extract of the leaves, which was then examined for enzyme inhibitory effectiveness against COX and 5-LOX enzymes. All fractions and CE were examined for antioxidant activity, and phenolic acids and flavonoids were identified using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The ethyl acetate fraction (EaF) had the greatest COX/5-LOX inhibitory impact among tested fractions.In vitro, COX activity studies showed that EaF and butanol fraction (BF) possess a higher COX-2 selectivity index than Indomethacin. Besides, the EaF exhibited the highest reduction potential among the control and the CE when tested for antioxidant activities. These findings support the traditional use of Melia azedarachas an anti-inflammatory drug, as the plant inhibited both COX/5-LOX and COX-2 with high selectivity.1.Introduction The most frequent chemical species that cause oxidative damage in the body are reactive oxygen species (ROS) like peroxides, superoxide, singlet oxygen, hydroxyl radical, hydrogen peroxides, and so on. These species produced in the body can cause various cardiovascular and other chronic diseases [1-3], neurodegenerative diseases [4,5], and inflammatory diseases [6,7]. Different inflammatory diseases are linked to oxidative stress (an imbalance between ROS and their removal by protective mechanisms such as antioxidants) [7-9]. Inflammatory mediators such as prostaglandins produced by the arachidonic acid pathway need to be reduced and inhibited as they mainly cause inflammation, pain, and fever in the body [10]. Currently, the symptomatic treatment of inflammation is mainly done by non-steroidal anti-inflammatory drugs (NSAIDs). However, the traditional NSAIDs used in COX reduction are non-selective, lead to gastrointestinal side effects, heart strokes and renal failure. On the other hand, the currently used selective COX-2 inhibitors like ‘coxibs' are reported for cardiovascular side effects [11]. In addition to COX-2, the increased level of leukotriene from the 5-LOX enzyme can result in bronchospasm [11]. In this regard, medicinal preparations that inhibit both Cyclooxygenase (COX-2) and 5-Lipoxygenase (5-LOX) pathways are superior to commonly used NSAIDs since they could produce a synergetic effect to achieve optimal anti-inflammatory activities [10,11]. This can be done by blocking the production of both leukotrienes and prostaglandins. Hence, indeed, there is a need for new, effective, and safe anti-inflammatory drugs with dual COX-2/5-LOX inhibition capacities, which may obliterate the side effects caused by the inhibition of COX-2 alone [12,13]. Melia azedarachL. (known commonly as “Chinaberry, bead tree, Bakain and Persian lilac") is a deciduous tree belonging to the Meliaceae family that is native to tropical Asia and Australia but has now spread throughout the world. [14-16]. The antioxidantand anti-inflammatory potential of Melia azedarachL. from various regions werereported by other groups [14,15, 17-21]. The plant with different species and some species of different cultivars markedly differin their phenolics, flavonoids and antioxidant activities [18,20]. The antioxidant and anti-inflammatory activities of plant extracts are closely linked to phytoconstituents like total phenolics and flavonoids [22-25]. There is no scientific report on the anti-inflammatory action and antioxidant potential of Taiwanese-originMelia azedarach. Therefore, the purpose of the current study is to investigate the dual COX-2 and 5-LOX inhibitor potential, antioxidant potential, quantification of key flavonoids and phenolic acids, as well as total phenolic and flavonoid content of Melia azedarach.

Novel Dual COX-2/5-LOX Inhibitory Activity by Chalcone Derivatives: A Safe and Efficacious Anti-inflammatory Agent.

Non-communicable diseases are chronic systemic inflammation in humans that occurs because of enhanced inflammatory mediators of the arachidonic acid cascade. We aimed to explore whether the lead chalcone compounds could exhibit anti-inflammatory activity via dual blockage of COX-2/5-LOX enzymes and their regulatory mechanism. RAW 264.7 macrophages were collected from NCC, Pune, for in-vitro experiments. The IC50 values of chalcone compounds C45 and C64 were calculated. RAW 264.7 macrophages were treated with C45 and C64 (10%, 5%, 2.5%, 0.125%, and 0.0625% concentration). The cell viability was carried out with an MTT assay. The COX-1, COX-2, 5-LOX, PGE2, and LTB4 levels were detected by ELISA-based kits. The in-vivo evaluation was carried out in Male Wistar rats (250-300 g, 7-8 weeks old) with acute and chronic anti-inflammatory models and histopathological studies on the stomach, liver, and kidney. The present study described the in-vitro and in-vivo biological evaluation of dual COX-2/5-LOX inhibitors in chalcone derivatives (C45 and C64) compounds showed the most effective COX-2 and 5-LOX inhibition with IC50 values 0.092 and 0.136 μM respectively. Simultaneously, compound C64 showed comparable selectivity towards COX-2 with a Selectivity Index (SI) of 68.43 compared to etoricoxib, with an SI of 89.32. In-vivo carrageenaninduced rat paw oedema activity, the compound C64 showed a significant reduction in oedema with 78.28% compared to indomethacin with 88.07% inhibition. Furthermore, cotton pelletinduced granuloma activity revealed that compound C64 significantly reduced 32.85% compared with standard 40.13% granuloma inhibition. The chalcone compound C64, (E)-1-(4-Amino-2-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)- prop-2-en-1-one was proved to be a potent and novel Dual COX-2/5-LOX inhibitor with improved gastric safety profiling.

Evaluation of the Interaction between Carvacrol and Thymol, Major Compounds of Ptychotis verticillata Essential Oil: Antioxidant, Anti-Inflammatory and Anticancer Activities against Breast Cancer Lines.

The objective of this study was to evaluate the antioxidant, anti-inflammatory, and anticancer properties of thymol, carvacrol, and their equimolar mixture. Antioxidant activities were assessed using the DPPH, ABTS, and ORAC methods. The thymol/carvacrol mixture exhibited significant synergism, surpassing the individual compounds and ascorbic acid in DPPH (IC50 = 43.82 ± 2.41 µg/mL) and ABTS (IC50 = 23.29 ± 0.71 µg/mL) assays. Anti-inflammatory activity was evaluated by inhibiting the 5-LOX, COX-1, and COX-2 enzymes. The equimolar mixture showed the strongest inhibition of 5-LOX (IC50 = 8.46 ± 0.92 µg/mL) and substantial inhibition of COX-1 (IC50 = 15.23 ± 2.34 µg/mL) and COX-2 (IC50 = 14.53 ± 2.42 µg/mL), indicating a synergistic effect. Anticancer activity was tested on MCF-7, MDA-MB-231, and MDA-MB-436 breast cancer cell lines using the MTT assay. The thymol/carvacrol mixture demonstrated superior cytotoxicity (IC50 = 0.92-1.70 µg/mL) and increased selectivity compared to cisplatin, with high selectivity indices (144.88-267.71). These results underscore the promising therapeutic potential of the thymol/carvacrol combination, particularly for its synergistic antioxidant, anti-inflammatory, and anticancer properties against breast cancer. This study paves the way for developing natural therapies against breast cancer and other conditions associated with oxidative stress and inflammation, leveraging the synergistic effects of natural compounds like thymol and carvacrol.

Therapeutic potential of sulfated glycosaminoglycan from seafood Asian green mussel (Perna viridis): Insights from an in vivo study

The Asian green mussel, Perna viridis, is widely consumed as seafood throughout the coastal regions of the Indo-Pacific area owing to its significant pharmaceutical and biomedical benefits. A pharmacologically active (1 → 3)/(1 → 4) linked sulfated glycosaminoglycan (PVP-2) with β-(1 → 3)-GlcNSp, and α-(1 → 4)-GlcAp units was isolated from P. viridis. PVP-2 (at 1–10 μg/mL) demonstrated a suppression of nitric oxide (NO) secretion in lipopolysaccharide-induced RAW 264.7 cells. Notably, PVP-2 at 5 μg/mL effectively restored NO levels (0.32 μg/mL) to homeostasis by downregulating excessive production (1.22 μg/mL). Furthermore, PVP-2 (at 22 mg/kg body weight) showed effective reduction of carrageenan-induced paw edema (82% inhibition at 5 h) in time dependent manner. This reduction was found to be comparable with standard treatment (87% inhibition of paw edema at 5 h). Treatment with PVP-2 (at 110 mg/kg body weight) resulted substantial inhibition of formalin-induced paw edema up to 10 days. At the end of the 10th day, the reduction in paw thickness was significantly greater (a four-fold increase) in the PVP-2 treated group compared to the formalin-induced group. Furthermore, PVP-2 displayed potent anti-inflammatory effect by attenuating cyclooxygenases (COX-1, 2) and 5-lipoxygenase (5-LOX) (IC50 < 2 mg/mL) enzymes with a higher selectivity index (IC50 COX-1/IC50 COX-2 ∼ 1.7) towards inducible pro-inflammatory COX-2. Therefore, inhibition of the NO production along with the reduction of paw edema, highlights the therapeutic potential of sulfated glycosaminoglycan from seafood Asian green mussel, as a naturally derived functional food to mitigate inflammation related disorders.

Interactions between Inhibitors and 5-Lipoxygenase: Insights from Gaussian Accelerated Molecular Dynamics and Markov State Models.

Inflammation is a protective stress response triggered by external stimuli, with 5-lipoxygenase (5LOX) playing a pivotal role as a potent mediator of the leukotriene (Lts) inflammatory pathway. Nordihydroguaiaretic acid (NDGA) functions as a natural orthosteric inhibitor of 5LOX, while 3-acetyl-11-keto-β-boswellic acid (AKBA) acts as a natural allosteric inhibitor targeting 5LOX. However, the precise mechanisms of inhibition have remained unclear. In this study, Gaussian accelerated molecular dynamics (GaMD) simulation was employed to elucidate the inhibitory mechanisms of NDGA and AKBA on 5LOX. It was found that the orthosteric inhibitor NDGA was tightly bound in the protein's active pocket, occupying the active site and inhibiting the catalytic activity of the 5LOX enzyme through competitive inhibition. The binding of the allosteric inhibitor AKBA induced significant changes at the distal active site, leading to a conformational shift of residues 168-173 from a loop to an α-helix and significant negative correlated motions between residues 285-290 and 375-400, reducing the distance between these segments. In the simulation, the volume of the active cavity in the stable conformation of the protein was reduced, hindering the substrate's entry into the active cavity and, thereby, inhibiting protein activity through allosteric effects. Ultimately, Markov state models (MSM) were used to identify and classify the metastable states of proteins, revealing the transition times between different conformational states. In summary, this study provides theoretical insights into the inhibition mechanisms of 5LOX by AKBA and NDGA, offering new perspectives for the development of novel inhibitors specifically targeting 5LOX, with potential implications for anti-inflammatory drug development.

An engineered lactate oxidase based electrochemical sensor for continuous detection of biomarker lactic acid in human sweat and serum

Lactate levels in humans reveal intensity and duration of exertion and provide a critical readout for the severity of life-threatening illnesses such as pediatric sepsis. Using the lactate oxidase enzyme (Lox) from Aerococcus viridians, we demonstrated its functionality for lactate electrochemical sensing in physiological fluids in a lab setting. The structure and dynamics of LOx were validated by crystallography, X-ray scattering, and hydroxyl radical protein footprinting. This provided a validated protein template for understanding and designing an enzyme-based electrochemical sensing elements. Using this template, LOx enzyme variants were generated and compared. Comparison of the variants demonstrates that one exhibits effective lactate sensing at significantly reduced operating voltages. Additionally, we demonstrate that the four hexahistidine-tags on each enzyme tetramer are sufficient for immobilization to create a durable, functional sensor, with no need for a covalent attachment, enabling self-immobilization and eliminating the need for additional immobilization steps. The functionality of the LOx enzyme variants was verified at physiological lactate concentrations in both human serum (0–4 mM) and artificial sweat (0–100 mM) using 3-electrode setups for analysis of the three variants in parallel. Accuracy of measurement in both artificial sweat and human serum were high. Employing a microfluidic flow cell, we successfully monitored varying lactate levels in physiological fluids continuously over a 2h period. Overall, this optimized LOx enzyme, which self-immobilizes onto gold sensing electrodes, facilitates efficient and reliable lactate detection and continuous monitoring at reduced operating voltages suitable for further development towards commercial use.

Anti-Inflammatory, Antidiabetic, and Antioxidant Properties of Extracts Prepared from Pinot Noir Grape Marc, Free and Incorporated in Porous Silica-Based Supports.

This study presents properties of hydroethanolic extracts prepared from Pinot Noir (PN) grape pomace through conventional, ultrasound-assisted or solvothermal extraction. The components of the extracts were identified by HPLC. The total content of polyphenols, flavonoids, anthocyanins, and condensed tannins, as well as antioxidant activity and α-glucosidase inhibitory activity of extracts were evaluated using UV-vis spectroscopy. All extracts were rich in phenolic compounds, proving a good radical scavenging activity. The extract obtained by conventional extraction at 80 °C showed the best α-glucosidase inhibitory activity close to that of (-)-epigallocatechin gallate. To improve the chemical stability of polyphenols, the chosen extract was incorporated in porous silica-based supports: amine functionalized silica (MCM-NH2), fucoidan-coated amine functionalized silica (MCM-NH2-Fuc), MCM-41, and diatomite. The PN extract exhibited moderate activity against Gram-positive S. aureus (MIC = 156.25 μg/mL) better than against Gram-negative E. coli (MIC = 312.5 μg/mL). The biocompatibility of PN extract, free and incorporated in MCM-NH2 and MCM-NH2-Fuc, was assessed on RAW 264.7 mouse macrophage cells, and the samples showcased a good cytocompatibility at 10 µg/mL concentration. At this concentration, PN and PN@MCM-NH2-Fuc reduced the inflammation by inhibiting NO production. The anti-inflammatory potential against COX and LOX enzymes of selected samples was evaluated and compared with that of Indomethacin and Zileuton, respectively. The best anti-inflammatory activity was observed when PN extract was loaded on MCM-NH2-Fuc support.

Phytochemical profiling and therapeutic potential of Baliospermum montanum: A promising inhibitor of COX-2/15-LOX enzymes and NO production in LPS-stimulated RAW 264.7 macrophages

IntroductionBaliospermum montanum (BM) is traditionally used to treat various ailments such as inflammation, rheumatoid arthritis, pain, etc. The root part of BM is well documented for its anti-inflammatory activity. However, the present study evaluates the multitargeted anti-inflammatory activity of the less explored BM leaf part. This is the first study investigating the COX-2, 15-LOX dual inhibiting potential and NO production inhibition of BM leaf extracts, fractions and compounds. MethodsThe BM leaf's water, ethanol, and hydroethanolic extracts were evaluated for anti-inflammatory activity by assessing the inhibition of COX-2, 15-LOX, and NO production. Additionally, the antioxidant potential of the extracts was determined through DPPH, ABTS, and FRAP assays. The most active ethanol extract was fractionated into 12 fractions and tested for anti-inflammatory, antioxidant, phenol and flavonoid content. Compounds were isolated from the most active fractions and identified using NMR techniques. The isolated compounds were checked for their anti-inflammatory activity. In addition, HPLC analysis of ethanol extract, fractions, and isolated compounds was carried out. ResultsThe ethanol extract exhibited higher inhibition of COX-2, 15-LOX enzymes and NO production (IC50 35.75 ± 1.95 μg/mL) in LPS-treated RAW 264.7 cells. It also showed higher antioxidant activity. Following the fractionation of the ethanol extract using different solvents, only the ethyl acetate fractions demonstrated inhibition of COX-2, 15-LOX enzymes, and NO production. The ethyl acetate and acetone fractions are excellent sources of antioxidants, phenolic and flavonoid compounds. An effort to find the active principles from these ethyl acetate fractions resulted in the isolation and identification of four potent anti-inflammatory agents, namely, (-)-epiafzelechin, afzelechin-(4α→8)-afzelechin, afzelechin-(4α→8)-epiafzelechin and β-sitosterol-d-glycoside. The first three compounds were reported for the first time from this plant and afzelechin-(4α→8)-afzelechin was found to be a potent inhibitor of the COX-2/15 LOX enzyme as well as NO production (IC50 = 5.9 ± 1.09 µM). ConclusionBased on these findings, we conclude that ethanol extract, fractions, and isolated compounds exert anti-inflammatory activity through multiple therapeutic mechanisms. The present study scientifically supports the traditional use of plant BM for treating inflammation and other related disorders.

In-vitro and molecular docking studies of plant secondary metabolites isolated from Hypericum oblongifolium as antibacterial agents and lipoxygenase (5-LOX) inhibitors

Natural Products are bases for modern pharmaceuticals as they are precursors for many synthetic drugs. People also use natural products directly in the form of herbal medicines due to their less harmful effects. This research project aimed to investigate the new pharmacological potential of natural products found in Hypericum oblongifolium. Five compounds were identified from chloroform and ethyl acetate soluble fractions of H. oblongifolium. These compounds were identified as 21-β-hydroxyursolic acid (1), β-sitosterol (2), 7,4′-dihydroxy-5,3′-dimethoxyisoflavone (3), α-D-glucopyranosyl-6′-O-hexadecanoate (4), Quercetin-3′-O-ß-D-glucopyranoside (5). When these compounds were tested for antibacterial potential compound 5 showed maximum inhibition against gram-positive Bacillus subtilis and was highly active against Escherichia coli gram-negative bacterium. Minimum inhibition was shown by compounds 2 and 3 against Shigella flexneri among gram-negative bacteria. Compound 5 also exhibited the least binding energy in the molecular modeling of these compounds against Escherichia coli proteins GlcN-6-P synthase (PDB ID: 1MOQ). Compound 5 also showed good inhibition of lipoxygenase (5-LOX) enzyme with an IC50 value of 30.1 ± 0.20 as it exhibited maximum hydrogen bondings and minimum energy when docked with LOX-5 (PDB ID 3V99) using a patch dock.

LC-MS/MS profiling of Tipuana tipu flower, HPLC-DAD quantification of its bioactive components, and interrelationships with antioxidant, and anti-inflammatory activity: in vitro and in silico approaches

BackgroundFabaceae plays a crucial role in African traditional medicine as a source of large number of important folk medication, agriculture and food plants. In a search of potential antioxidant and anti-inflammatory candidates derived from locally cultivated plants, the flowers of Tipuana tipu (Benth.) Lillo growing in Egypt were subjected to extensive biological and phytochemical studies. The impact of the extraction technique on the estimated biological activities was investigated.MethodsThe flowers were extracted using different solvents (aqueous, methanol, water/methanol (1:1), methanol/methylene chloride (1:1), and methylene chloride). The different extracts were subjected to antioxidant (DPPH, ABTS, and FRAP) and anti-inflammatory (COX-2 and 5-LOX) assays. The methanol extract was assessed for its inhibitory activity against iNOS, NO production, and pro-inflammatory cytokines (NF-KB, TNF-R2, TNF-α, IL-1β, and IL-6) in LPS-activated RAW 264.7 macrophages. The composition-activity relationship of the active methanol extract was further investigated using a comprehensive LC–QTOF-MS/MS analysis. The major identified phenolic compounds were further quantified using HPLC-DAD technique. The affinity of representative compounds to iNOS, COX-2, and 5-LOX target active sites was investigated using molecular docking and molecular dynamics simulations.ResultsThe methanol extract exhibited the highest radical scavenging capacity and enzyme inhibitory activities against COX-2 and 5-LOX enzymes with IC50 values of 10.6 ± 0.4 and 14.4 ± 1.0 µg/mL, respectively. It also inhibited iNOS enzyme activity, suppressed NO production, and decreased the secretion of pro-inflammatory cytokines. In total, 62 compounds were identified in the extract including flavonoids, coumarins, organic, phenolic, and fatty acids. Among them 18 phenolic compounds were quantified by HPLC-DAD. The highest docking scores were achieved by kaempferol-3-glucoside and orientin. Additionally, molecular dynamics simulations supported the docking findings.ConclusionThe flower could be considered a potentially valuable component in herbal medicines owing to its unique composition and promising bioactivities. These findings encourage increased propagation of T. tipu or even tissue culturing of its flowers for bioprospecting of novel anti-inflammatory drugs. Such applications could be adopted as future approaches that benefit the biomedical field.

The Composition of Volatiles and the Role of Non-Traditional LOX on Target Metabolites in Virgin Olive Oil from Autochthonous Dalmatian Cultivars.

The lipoxygenase pathway has a significant influence on the composition of the volatile components of virgin olive oil (VOO). In this work, the influence of the maturity index (MI) on the activity of the lipoxygenase enzyme (LOX) in the fruits of the autochthonous Dalmatian olive cultivars Oblica, Levantinka and Lastovka was studied. The analysis of the primary oxidation products of linoleic acid in the studied cultivars showed that LOX synthesises a mixture of 9- and 13-hydroperoxides of octadecenoic acid in a ratio of about 1:2, which makes it a non-traditional plant LOX. By processing the fruits of MI~3, we obtained VOOs with the highest concentration of desirable C6 volatile compounds among the cultivars studied. We confirmed a positive correlation between MI, the enzyme activity LOX and the concentration of hexyl acetate and hexanol in cultivars Oblica and Lastovka, while no positive correlation with hexanol was observed in the cultivar Levantinka. A significant negative correlation was found between total phenolic compounds in VOO and LOX enzyme activity, followed by an increase in the MI of fruits. This article contributes to the selection of the optimal harvest time for the production of VOOs with the desired aromatic properties and to the knowledge of the varietal characteristics of VOOs.

RETRACTED ARTICLE: Bio synthesis, comprehensive characterization, and multifaceted therapeutic applications of BSA-Resveratrol coated platinum nanoparticles

This study examines the manufacturing, characterization, and biological evaluation of platinum nanoparticles, which were synthesized by Enterobacter cloacae and coated with Bovine Serum Albumin (BSA) and Resveratrol (RSV). The formation of PtNPs was confirmed with the change of color from dark yellow to black, which was due to the bioreduction of platinum chloride by E. cloacae. BSA and RSV functionalization enhanced these nanoparticles' biocompatibility and therapeutic potential. TGA, SEM, XRD, and FTIR were employed for characterization, where PtNPs and drug conjugation-related functional groups were studied by FTIR. XRD confirmed the crystalline nature of PtNPs and Pt-BSA-RSV NPs, while TGA and SEM showed thermal stability and post-drug coating morphological changes. Designed composite was also found to be biocompatible in nature in hemolytic testing, indicating their potential in Biomedical applications. After confirmation of PtNPs based nanocaompsite synthesis, they were examined for anti-bacterial, anti-oxidant, anti-inflammatory, and anti-cancer properties. Pt-BSA-RSV NPs showed higher concentration-dependent DPPH scavenging activity, which measured antioxidant capability. Enzyme inhibition tests demonstrated considerable anti-inflammatory activity against COX-2 and 15-LOX enzymes. In in vitro anticancer studies, Pt-BSA-RSV NPs effectively killed human ovarian cancer cells. This phenomenon was demonstrated to be facilitated by the acidic environment of cancer, as the drug release assay confirmed the release of RSV from the NP formulation in the acidic environment. Finally, Molecular docking also demonstrated that RSV has strong potential as an anti-oxidant, antibacterial, anti-inflammatory, and anticancer agent. Overall, in silico and in vitro investigations in the current study showed good medicinal applications for designed nanocomposites, however, further in-vivo experiments must be conducted to validate our findings.

Modification of 4-(4-chlorothiophen-2-yl)thiazol-2-amine derivatives for the treatment of analgesia and inflammation: synthesis and in vitro, in vivo, and in silico studies.

Inflammation is a protective response to a variety of infectious agents. To develop a new anti-inflammatory drug, we explored a pharmacologically important thiazole scaffold in this study. In a multi-step synthetic approach, we synthesized seven new thiazole derivatives (5a-5g). Initially, we examined the in vitro anti-inflammatory potentials of our compounds using COX-1, COX-2, and 5-LOX enzyme assays. After in vitro confirmation, the potential compounds were subjected to in vivo analgesic and anti-inflammatory studies. The hot plate method was used for analgesia, and carrageenan-induced inflammation was also assayed. Overall, all our compounds proved to be potent inhibitors of COX-2 compared to celecoxib (IC50 0.05μM), exhibiting IC50 values in the range of 0.76-9.01μM .Compounds 5b, 5d, and 5e were dominant and selective COX-2 inhibitors with the lowest IC50 values and selectivity index (SI) values of 42, 112, and 124, respectively. Similarly, in the COX-1 assay, our compounds were relatively less potent but still encouraging. Standard aspirin exhibited an IC50 value of 15.32μM. In the 5-LOX results, once again, compounds 5d and 5e were dominant with IC50 values of 23.08 and 38.46μM, respectively. Standard zileuton exhibited an IC50 value of 11.00μM. Based on the COX/LOX and SI potencies, the compounds 5d and 5e were subjected to in vivo analgesic and anti-inflammatory studies. Compounds 5d and 5e at concentrations of 5, 10, and 20mg/kg body weight were significant in animal models. Furthermore, we explored the potential role of compounds 5d and 5e in various phlogistic agents. Similarly, both compounds 5d and 5e were also significantly potent in the anti-nociceptive assay. The molecular docking interactions of these two compounds with the target proteins of COX and LOX further strengthened their potential for use in COX/LOX pathway inhibitions.