Molecular Interactions and Mechanisms of COVID-19 Inhibition 2.0.

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Scavenging of Superoxide in Aprotic Solvents of Four Isoflavones That Mimic Superoxide Dismutase.

Isoflavones are plant-derived natural products commonly found in legumes that show a large spectrum of biomedical activities. A common antidiabetic remedy in traditional Chinese medicine, Astragalus trimestris L. contains the isoflavone formononetin (FMNT). Literature reports show that FMNT can increase insulin sensitivity and potentially target the peroxisome proliferator-activated receptor gamma, PPARγ, as a partial agonist. PPARγ is highly relevant for diabetes control and plays a major role in Type 2 diabetes mellitus development. In this study, we evaluate the biological role of FMNT, and three related isoflavones, genistein, daidzein and biochanin A, using several computational and experimental procedures. Our results reveal the FMNT X-ray crystal structure has strong intermolecular hydrogen bonding and stacking interactions which are useful for antioxidant action. Cyclovoltammetry rotating ring disk electrode (RRDE) measurements show that all four isoflavones behave in a similar manner when scavenging the superoxide radical. DFT calculations conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving hydrogen capture of ring-A H7(hydroxyl) as well as the π-π (polyphenol-superoxide) scavenging activity. These results suggest the possibility of their mimicking superoxide dismutase (SOD) action and help explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate O2•- to H2O2 plus O2 through metal ion redox chemistry whereas these polyphenolic compounds do so through suitable hydrogen bonding and stacking intermolecular interactions. Additionally, docking calculations suggest FMNT can be a partial agonist of the PPARγ domain. Overall, our work confirms the efficacy in combining multidisciplinary approaches to provide insight into the mechanism of action of small molecule polyphenol antioxidants. Our findings promote the further exploration of other natural products, including those known to be effective in traditional Chinese medicine for potential drug design in diabetes research.

Maytenus octogona Superoxide Scavenging and Anti-Inflammatory Caspase-1 Inhibition Study Using Cyclic Voltammetry and Computational Docking Techniques.

The relationship between oxidative stress and inflammation is well known, and exogenous antioxidants, primarily phytochemical natural products, may assist the body's endogenous defense systems in preventing diseases due to excessive inflammation. In this study, we evaluated the antioxidant properties of ethnomedicines from Peru that exhibit anti-inflammatory activity by measuring the superoxide scavenging activity of ethanol extracts of Maytenus octogona aerial parts using hydrodynamic voltammetry at a rotating ring-disk electrode (RRDE). The chemical compositions of these extracts are known and the interactions of three methide-quinone compounds found in Maytenus octogona with caspase-1 were analyzed using computational docking studies. Caspase-1 is a critical enzyme triggered during the activation of the inflammasome and its actions are associated with excessive release of cytokines. The most important amino acid involved in active site caspase-1 inhibition is Arg341 and, through docking calculations, we see that this amino acid is stabilized by interactions with the three potential methide-quinone Maytenus octogona inhibitors, hydroxytingenone, tingenone, and pristimerin. These findings were also confirmed after more rigorous molecular dynamics calculations. It is worth noting that, in these three compounds, the methide-quinone carbonyl oxygen is the preferred hydrogen bond acceptor site, although tingenone's other carbonyl group also shows a similar binding energy preference. The results of these calculations and cyclovoltammetry studies support the effectiveness and use of anti-inflammatory ethnopharmacological ethanol extract of Maytenus octogona (L'Héritier) DC.

Ruthenium(II)-Arene Curcuminoid Complexes as Photosensitizer Agents for Antineoplastic and Antimicrobial Photodynamic Therapy: In Vitro and In Vivo Insights.

Photodynamic therapy (PDT) is an anticancer/antibacterial strategy in which photosensitizers (PSs), light, and molecular oxygen generate reactive oxygen species and induce cell death. PDT presents greater selectivity towards tumor cells than conventional chemotherapy; however, PSs have limitations that have prompted the search for new molecules featuring more favorable chemical-physical characteristics. Curcumin and its derivatives have been used in PDT. However, low water solubility, rapid metabolism, interference with other drugs, and low stability limit curcumin use. Chemical modifications have been proposed to improve curcumin activity, and metal-based PSs, especially ruthenium(II) complexes, have attracted considerable attention. This study aimed to characterize six Ru(II)-arene curcuminoids for anticancer and/or antibacterial PDT. The hydrophilicity, photodegradation rates, and singlet oxygen generation of the compounds were evaluated. The photodynamic effects on human colorectal cancer cell lines were also assessed, along with the ability of the compounds to induce ROS production, apoptotic, necrotic, and/or autophagic cell death. Overall, our encouraging results indicate that the Ru(II)-arene curcuminoid derivatives are worthy of further investigation and could represent an interesting option for cancer PDT. Additionally, the lack of significant in vivo toxicity on the larvae of Galleria mellonella is an important finding. Finally, the photoantimicrobial activity of HCurc I against Gram-positive bacteria is indeed promising.

Aromatic Polyphenol π-π Interactions with Superoxide Radicals Contribute to Radical Scavenging and Can Make Polyphenols Mimic Superoxide Dismutase Activity.

Polyphenols are valuable natural antioxidants present in our diet that likely mitigate aging effects, neurodegenerative conditions, and other diseases. However, because of their poor absorption in the gut and consequent low concentration in biological fluids (µM range), reservations about polyphenol antioxidant efficiency have been raised. In this review, it is shown that after scavenging superoxide radicals, coumarin, chalcone, and flavonoid polyphenols can reform themselves, becoming ready for additional cycles of scavenging, similar to the catalytic cycle in superoxide dismutase (SOD) action. The π-π interaction between one polyphenol ring and superoxide is associated with oxidation of the latter due to transfer of its unpaired electron to a polyphenolic aromatic ring, and consequent formation of a molecule of O2 (one product of SOD action). Mechanistically, it is very difficult to establish if this π-π interaction proceeds before or after the most common mode of scavenging superoxide, e.g., abstraction of an aromatic polyphenol H(hydroxyl), which then is used to form H2O2 (the other molecule produced by SOD action). At the end of this cycle of superoxide scavenging, 4-methyl-7,8-di-hydroxy-coumarin and the flavonoid galangin reform themselves. An alternative mechanistic pathway by galangin forms the η-(H2O2)-galangin-η-O2 complex that includes additional H2O2 and O2 molecules. Another mode of action is seen with the chalcone butein, in which the polyphenol system incorporates a molecule of O2, e.g., a η-O2-butein complex is formed, ready for additional scavenging. Of the several families of polyphenols analyzed in this review, only butein was able to circumvent an initial π-π interaction, directing the superoxide towards H(hydroxyl) in position 4, e.g., acting as a typical polyphenol scavenger of superoxide. This fact did not impede an additional superoxide to later react with the aromatic ring in π-π fashion. It is concluded that by mimicking SOD enzyme action, the low concentration of polyphenols in biological fluids is not a limiting factor for effective scavenging of superoxide.

Comments about the IJMS Special Issue: Molecular Interactions and Mechanisms of COVID-19 Inhibition.

The unprecedented COVID-19 pandemic showed up during the latter part of 2019 in Wuhan, China [...].

Mechanism of Caspase-1 Inhibition by Four Anti-inflammatory Drugs Used in COVID-19 Treatment.

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.

X-ray Structure Determination, Antioxidant Voltammetry Studies of Butein and 2',4'-Dihydroxy-3,4-dimethoxychalcone. Computational Studies of 4 Structurally Related 2',4'-diOH Chalcones to Examine Their Antimalarial Activity by Binding to Falcipain-2.

Malaria is a huge global health burden with resistance to currently available medicines resulting in the search for newer antimalarial compounds from traditional medicinal plants in malaria-endemic regions. Previous studies on two chalcones, homobutein and 5-prenylbutein, present in E. abyssinica, have shown moderate antiplasmodial activity. Here, we describe results from experimental and computational investigations of four structurally related chalcones, butein, 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDM), homobutein and 5-prenylbutein to elucidate possible molecular mechanisms by which these compounds clear malaria parasites. The crystal structures of butein and DHDM show that butein engages in more hydrogen bonding and consequently, more intermolecular interactions than DHDM. Rotating ring-disk electrode (RRDE) voltammetry results show that butein has a higher antioxidant activity towards the superoxide radical anion compared to DHDM. Computational docking experiments were conducted to examine the inhibitory potential of all four compounds on falcipain-2, a cysteine protease that is involved in the degradation of hemoglobin in plasmodium-infected red blood cells of the host. Overall, this work suggests butein as a better antimalarial compound due to its structural features which allow it to have greater intermolecular interactions, higher antioxidant activity and to create a covalent complex at the active site of falcipain-2.

Interrelated Mechanism by Which the Methide Quinone Celastrol, Obtained from the Roots of Tripterygium wilfordii, Inhibits Main Protease 3CLpro of COVID-19 and Acts as Superoxide Radical Scavenger.

We describe the potential anti coronavirus disease 2019 (COVID-19) action of the methide quinone inhibitor, celastrol. The related methide quinone dexamethasone is, so far, among COVID-19 medications perhaps the most effective drug for patients with severe symptoms. We observe a parallel redox biology behavior between the antioxidant action of celastrol when scavenging the superoxide radical, and the adduct formation of celastrol with the main COVID-19 protease. The related molecular mechanism is envisioned using molecular mechanics and dynamics calculations. It proposes a covalent bond between the S(Cys145) amino acid thiolate and the celastrol A ring, assisted by proton transfers by His164 and His41 amino acids, and a π interaction from Met49 to the celastrol B ring. Specifically, celastrol possesses two moieties that are able to independently scavenge the superoxide radical: the carboxylic framework located at ring E, and the methide-quinone ring A. The latter captures the superoxide electron, releasing molecular oxygen, and is the feature of interest that correlates with the mechanism of COVID-19 inhibition. This unusual scavenging of the superoxide radical is described using density functional theory (DFT) methods, and is supported experimentally by cyclic voltammetry and X-ray diffraction.

The Grapefruit Effect: Interaction between Cytochrome P450 and Coumarin Food Components, Bergamottin, Fraxidin and Osthole. X-ray Crystal Structure and DFT Studies.

Coumarins are plant-derived secondary metabolites. The crystal structure of three coumarins-bergamottin, osthole and fraxidin-are described and we analyze intermolecular interactions and their role in crystal formation. Bergamottin is a furanocoumarin found in citrus plants, which is a strong inhibitor of the principal human metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). The crystal structure determinations of three coumarins give us the geometrical parameters and reveal the parallel-displaced π-π stacking and hydrogen bonding intermolecular interactions used for molecular assembly in the crystal structure. A quite strong (less than 3.4 Å) stacking interaction of bergamottin appears to be a determining feature that distinguishes it from other coumarins studied in this work. Our DFT computational studies on the three natural products of the same coumarin family docked into the active site of CYP3A4 (PDB 4D78) show different behavior for these coumarins at the active site. When the substrate is bergamottin, the importance of π-π stacking and hydrogen bonding, which can anchor the substrate in place, appears fundamental. In contrast, fraxidin and osthole show carbonyl coordination to iron. Our docking calculations show that the bergamottin tendency towards π-π stacking is important and likely influences its interactions with the heme group of CYP3A4.

X-ray crystal structures, density functional theory and docking on deacetylase enzyme for antiproliferative activity of hispolon derivatives on HCT116 colon cancer.

The antiproliferative action of hispolon derivatives is stronger than that of related curcumin against several tumor cell lines. Hispolon size, smaller than curcumin, fits better than curcumin into the active site of HDAC6, an enzyme involved in deacetylation of lysine residues. HDACs are considered potential targets for tumor drug discovery and hydroxamates are known inhibitors of HDACs. One of them, SAHA (Vorinostat) is used in clinical studies. Investigations into possible mechanisms for hispolon derivatives active against the HCT116 colon tumor cell line are done after examining the structural results obtained from hispolon X-ray crystal structures as well as performing associated computational docking and Density Functional Theory techniques on HDAC6. These studies show preference for the HDAC6 active site by chelating the Zn center, in contrast with other ineffective hispolon derivatives, that establish only a single bond to the metal center. Structure activity relationships make clear that hydrogenation of the hispolon bridge also leads to single bond (non chelate) hispolon-Zn binding, and consistently nullifies the antiproliferative action against HCT116 tumor.

X-Ray Crystal Structure of Embelin and Its DFT Scavenging of Superoxide Radical.

Embelin is a phytochemical component of tropical plants that have a long history of being used in ethnic pharmacology in various parts of the world, including Ayurdvedic and Chinese medicinal texts. Many modern studies confirm its promise as a medicinal compound. The X-ray crystal structure determination of embelin shows a remarkably ordered alkyl chain and particularly strong pi-pi interactions for a nonaromatic system. The molecule has a torsion angle of 67° between the ring and the alkyl chain of the molecule and differs markedly from that seen when embelin is embedded in the plasminogen activator inhibitor-1 (PAI-1) binding site (almost planar-with about 10° torsion angle). This suggests that embelin's flexible structural skeleton can be useful in biological environment. Apart from this, its many biological activities likely depend on embelin's hydrophobic nonpolar tail that allows a variety of interactions. Computationally, we evaluated embelin's sequestering ability toward the superoxide radical and see that embelin executes this reaction in a novel manner. Namely, as shown by our DFT calculations, instead of releasing a H atom to the superoxide radical to form the anionic species O2 H- , embelin prefers to accept an electron from the superoxide radical, which then transforms into molecular oxygen, O2 . © 2017 Wiley Periodicals, Inc.

Regiospecifically Fluorinated Polycyclic Aromatic Hydrocarbons via Julia-Kocienski Olefination and Oxidative Photocyclization. Effect of Fluorine Atom Substitution on Molecular Shape.

A modular synthesis of regiospecifically fluorinated polycyclic aromatic hydrocarbons (PAHs) is described. 1,2-Diarylfluoroalkenes, synthesized via Julia-Kocienski olefination (70-99% yields), were converted to isomeric 5- and 6-fluorobenzo[c]phenanthrene, 5-and 6-fluorochrysene, and 9- and 10-benzo[g]chrysene (66-83% yields) by oxidative photocyclization. Photocyclization to 6-fluorochrysene proceeded more slowly than conversion of 1-styrylnaphthalene to chrysene. Higher fluoroalkene dilution led to a more rapid cyclization. Therefore, photocyclizations were performed at higher dilutions. To evaluate the effect of fluorine atom on molecular shapes, X-ray data for 5- and 6-fluorobenzo[c]phenanthrene, 6-fluorochrysene, 9- and 10-fluorobenzo[g]chrysene, and unfluorinated chrysene as well as benzo[g]chrysene were obtained and compared. The fluorine atom caused a small deviation from planarity in the chrysene series and decreased nonplanarity in the benzo[c]phenanthrene derivatives, but its influence was most pronounced in the benzo[g]chrysene series. A remarkable flattening of the molecule was observed in 9-fluorobenzo[g]chrysene, where the short 2.055 Å interatomic distance between bay-region F-9 and H-8, downfield shift of H-8, and a 26.1 Hz coupling between F-9 and C-8 indicate a possible F-9···H-8 hydrogen bond. In addition, in 9-fluorobenzo[g]chrysene, the stacking distance is short at 3.365 Å and there is an additional interaction between the C-11-H and C-10a of a nearby molecule that is almost perpendicular.

MicroRNAs expression patterns in the response of poplar woody root to bending stress.

MAIN CONCLUSION: The paper reports for the first time, in poplar woody root, the expression of five mechanically-responsive miRNAs. The observed highly complex expression pattern of these miRNAs in the bent root suggest that their expression is not only regulated by tension and compression forces highlighting their role in several important processes, i.e., lateral root formation, lignin deposition, and response to bending stress. Mechanical stress is one of the major abiotic stresses significantly affecting plant stability, growth, survival, and reproduction. Plants have developed complex machineries to detect mechanical perturbations and to improve their anchorage. MicroRNAs (miRNAs), small non-coding RNAs (18-24 nucleotides long), have been shown to regulate various stress-responsive genes, proteins and transcription factors, and play a crucial role in counteracting adverse conditions. Several mechanical stress-responsive miRNAs have been identified in the stem of Populus trichocarpa plants subjected to bending stress. However, despite the pivotal role of woody roots in plant anchorage, molecular mechanisms regulating poplar woody root responses to mechanical stress have still been little investigated. In the present paper, we investigate the spatial and temporal expression pattern of five mechanically-responsive miRNAs in three regions of bent poplar woody taproot and unstressed controls by quantitative RT-PCR analysis. Alignment of the cloned and sequenced amplified fragments confirmed that their nucleotide sequences are homologous to the mechanically-responsive miRNAs identified in bent poplar stem. Computational analysis identified putative target genes for each miRNA in the poplar genome. Additional miRNA target sites were found in several mechanical stress-related factors previously identified in poplar root and a subset of these was further analyzed for expression at the mRNA or protein level. Integrating the results of miRNAs expression patterns and target gene functions with our previous morphological and proteomic data, we concluded that the five miRNAs play crucial regulatory roles in reaction woody formation and lateral root development in mechanically-stressed poplar taproot.

Temporal analysis of poplar woody root response to bending stress.

Temperate perennial woody plants use different environmental signals to coordinate their growth and development in relation to seasonal changes. Preliminary evidences suggest that, even during dormancy, plants maintain effective metabolic activities and molecular mechanisms ensuring them an eventual recording of mechanical loads during winter times. Despite their great importance for productivity and survival, plant biology investigations have poorly characterized the root growth cycle and its response to environmental stresses. In this study, we describe the proteomic changes occurring over the time in poplar root either in the absence or in response to a bending stress; corresponding expression of cell cycle regulator and auxin transporter genes was also evaluated by reverse transcription polymerase chain reaction analysis. Our results confirm previous evidences on the effect of the bending stress on the anticipation of root growth resumption, providing additional insights on a temporal modulation of various plant metabolic processes involved in dormancy break, growth resumption and stress response in the bent root; these events seem related to the differential compression and tension force distribution occurring over the plant taproot.

Synthesis, characterization, and antitumor activity of water-soluble (arene)ruthenium(II) derivatives of 1,3-dimethyl-4-acylpyrazolon-5-ato ligands. First example of Ru(arene)(ligand) antitumor species involving simultaneous Ru-N7(guanine) bonding and ligand intercalation to DNA.

We report on the synthesis of novel water-soluble [(arene)Ru(II)(Q)Cl] and [(arene)Ru(II)(Q)(X)]BF4 compounds (arene = p-cymene, benzene, hexamethylbenzene; HQ = 1,3-dimethyl-4-R-(C═O)-5-pyrazolone, HQ(Me), R = methyl, HQ(Ph), R = phenyl, HQ(Naph), R = naphthyl; X = H2O, 9-ethylguanine), and their in vitro antitumor activity toward the cell lines MCF7 (HTB-22, human breast adenocarcinoma), HCT116 (CCL-247, human colorectal carcinoma), A2780 (human ovarian carcinoma), A549 (CCL-185, human lung carcinoma), and U87 MG (HTB-1, human glioblastoma). The X-ray crystal structures of two complexes were determined. One of them, {chlorido-(p-cymene)-[(1,3-dimethyl-4-(1-naphthoyl)-pyrazolon-5-ato]ruthenium(II)}, was also studied with density functional theory methods and was selected for docking on a DNA octamer showing intercalation between DNA bases by the naphthyl moiety and for Ru-N7(guanine) bonding.

Halogen bonding in (Z)-2-iodocinnamaldehyde.

Based on the bulkiness of the iodine atom, a non-planar conformation was expected for the title compound. Instead, its molecular structure is planar, as experimentally determined using single crystal X-ray diffraction, and confirmed theoretically by DFT calculations on the single molecule and the halogen pair paired molecules, therefore ruling out crystal packing forces as a principal factor leading to planarity. Indeed, planarity is ascribed to the carbonyl double bond, as when this bond is saturated on forming the related alcohol derivative, the molecule loses planarity. The X-ray molecular structure shows an intermolecular separation between the iodine and the oxygen of the carbonyl shorter than the corresponding van der Waals distance suggesting a weak halogen bond interaction. DFT minimization of this 2-molecule arrangement shows the iodine--oxygen distance much shorter than that observed in the crystal interaction and confirming its stronger halogen bond nature. A trend between increasing I•••O(carbonyl) separation and decreasing C-I•••O(carbonyl) angle is demonstrated, further confirming the existence of a halogen bond.

Azadiradione, a Component of Neem Oil, Behaves as a Superoxide Dismutase Mimic When Scavenging the Superoxide Radical, as Shown Using DFT and Hydrodynamic Voltammetry.

The neem tree, Azadirachta indica, belongs to the Meliaceae family, and its use in the treatment of medical disorders from ancient times to the present in the traditional medical practices of Asia, Africa and the Middle East is well-documented. Neem oil, extracted from the seeds of the fruit, is widely used, with promising medicinal benefits. Azadiradione, a principal antioxidant component of the seeds of A. indica, is known to reduce oxidative stress and has anti-inflammatory effects. To directly measure the antioxidant ability of neem oil, we used Rotating Ring Disk Electrode (RRDE) hydrodynamic voltammetry to quantify how it can scavenge superoxide radical anions. The results of these experiments show that neem oil is approximately 26 times stronger than other natural products, such as olive oil, propolis and black seed oil, which were previously measured using this method. Next, computational Density Functional Theory (DFT) methods were used to arrive at a mechanism for the scavenging of superoxide radical anions with azadiradione. Our work indicates that azadiradione is an effective antioxidant and, according to our DFT study, its scavenging of the superoxide radical anion occurs through a reaction mechanism in which azadiradione mimics the antioxidant action of superoxide dismutase (SOD). In this mechanism, analogous to the SOD enzymatic reaction, azadiradione is regenerated, along with the production of two products: hydrogen peroxide and molecular oxygen. This antioxidant process provides an explanation for azadiradione's more general and protective biochemical effects.

Antioxidant Properties of Thymoquinone, Thymohydroquinone and Black Cumin (Nigella sativa L.) Seed Oil: Scavenging of Superoxide Radical Studied Using Cyclic Voltammetry, DFT and Single Crystal X-ray Diffraction.

Black cumin seeds and seed oil have long been used in traditional foods and medicine in South Asian, Middle Eastern and Mediterranean countries and are valuable flavor ingredients. An important ingredient of black cumin is the small molecule thymoquinone (TQ), which manifests low toxicity and potential therapeutic activity against a wide number of diseases including diabetes, cancer and neurodegenerative disorders. In this study, the antioxidant activities of black seed oil, TQ and a related molecule found in black cumin, thymohydroquinone (THQ), were measured using a direct electrochemical method to experimentally evaluate their superoxide scavenging action. TQ and the black seed oil showed good superoxide scavenging ability, while THQ did not. Density Functional Theory (DFT) computational methods were applied to arrive at a chemical mechanism describing these results, and confirmed the experimental Rotating Ring Disk Electrode (RRDE) findings that superoxide oxidation to O2 by TQ is feasible, in contrast with THQ, which does not scavenge superoxide. Additionally, a thorough inquiry into the unusual cyclic voltammetry pattern exhibited by TQ was studied and was associated with formation of a 1:1 TQ-superoxide radical species, [TQ-O2]-•. DFT calculations reveal this radical species to be involved in the π-π mechanism describing TQ reactivity with superoxide. The crystal structures of TQ and THQ were analyzed, and the experimental data reveal the presence of stacking intermolecular interactions that can be associated with formation of the radical species, [TQ-O2]-•. All three of these methods were essential for us to arrive at a chemical mechanism that explains TQ antioxidant activity, that incorporates intermolecular features found in the crystal structure and which correlates with the measured superoxide scavenging activity.

Examining the Antioxidant and Superoxide Radical Scavenging Activity of Anise, (Pimpinella anisum L. Seeds), Esculetin, and 4-Methyl-Esculetin Using X-ray Diffraction, Hydrodynamic Voltammetry and DFT Methods.

Pimpinella anisum L., or anise, is a plant that, besides its nutritional value, has been used in traditional medical practices and described in many cultures in the Mediterranean region. A possible reason for anise's therapeutic value is that it contains coumarins, which are known to have many biomedical and antioxidant properties. HPLC analysis in our laboratory of the anise extract shows the presence of the coumarin esculetin. We used a hydrodynamic voltammetry rotating ring-disk electrode (RRDE) method to measure the superoxide scavenging abilities of anise seeds and esculetin, which has marked scavenging activity. A related coumarin, 4-methyl-esculetin, also showed strong antioxidant activity as measured by RRDE. Moreover, this study includes the X-ray crystal structure of esculetin and 4-methyl-esculetin, which reveal the H-bond and the stacking intermolecular interactions of the two coumarins. Coordinates of esculetin crystal structure were used to perform a DFT study to arrive at the mechanism of superoxide scavenging. Besides performing a H(hydroxyl) abstraction in esculetin position 6 by superoxide, the scavenging also includes the presence of a second superoxide radical in a π-π approach. Both rings of esculetin were explored for this attack, but only the pyrone ring was effective. As a result, one product of esculetin scavenging is H2O2 formation, while the second superoxide remains π-π trapped within the pyrone ring to form an esculetin-η-O2 complex. Comparison with other coumarins shows that subtle structural differences in the coumarin framework can imply marked differences in scavenging. For instance, when the catechol moiety of esculetin (position 6,7) is shifted to position 7,8 in 4-methyl-7,8-dihydroxy coumarin, that coumarin shows a superoxide dismutase action, which, beside H2O2 formation, includes the formation and elimination of a molecule of O2. This is in contrast with the products formed through esculetin superoxide scavenging, where a second added superoxide remains trapped, and forms an esculetin-η-O2 complex.