Chemistry, Biological Activities, and Pharmacological Properties of Gastrodin: Mechanism Insights.

Gastrodin, a bioactive compound derived from the rhizome of the orchid Gastrodia elata, exhibits a diverse range of biological activities. With documented neuroprotective, anti-inflammatory, antioxidant, anti-apoptotic, and anti-tumor effects, gastrodin stands out as a multifaceted therapeutic agent. Notably, it has demonstrated efficacy in protecting against neuronal damage and enhancing cognitive function in animal models of Alzheimer's disease, Parkinson's disease, and cerebral ischemia. Additionally, gastrodin showcases immunomodulatory effects by mitigating inflammation and suppressing the expression of inflammatory cytokines. Its cytotoxic activity involves the inhibition of angiogenesis, suppression of tumor growth, and induction of apoptosis. This comprehensive review seeks to elucidate the myriad potential effects of Gastrodin, delving into the intricate molecular mechanisms underpinning its pharmacological properties. The findings underscore the therapeutic potential of gastrodin in addressing various conditions linked to neuroinflammation and cancer.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Cooperative electrocatalytic alcohol oxidation with electron-proton-transfer mediators.

The electrochemical oxidation of alcohols is a major focus of energy and chemical conversion efforts, with potential applications ranging from fuel cells to biomass utilization and fine-chemical synthesis. Small-molecule electrocatalysts for processes of this type are promising targets for further development, as demonstrated by recent advances in nickel catalysts for electrochemical production and oxidation of hydrogen. Complexes with tethered amines that resemble the active site of hydrogenases have been shown both to catalyse hydrogen production (from protons and electrons) with rates far exceeding those of such enzymes and to mediate reversible electrocatalytic hydrogen production and oxidation with enzyme-like performance. Progress in electrocatalytic alcohol oxidation has been more modest. Nickel complexes similar to those used for hydrogen oxidation have been shown to mediate efficient electrochemical oxidation of benzyl alcohol, with a turnover frequency of 2.1 per second. These compounds exhibit poor reactivity with ethanol and methanol, however. Organic nitroxyls, such as TEMPO (2,2,6,6-tetramethyl-1-piperidine N-oxyl), are the most widely studied electrocatalysts for alcohol oxidation. These catalysts exhibit good activity (1­2 turnovers per second) with a wide range of alcohols and have great promise for electro-organic synthesis. Their use in energy-conversion applications, however, is limited by the high electrode potentials required to generate the reactive oxoammonium species. Here we report (2,2'-bipyridine)Cu/nitroxyl co-catalyst systems for electrochemical alcohol oxidation that proceed with much faster rates, while operating at an electrode potential a half-volt lower than that used for the TEMPO-only process. The (2,2'-bipyridine)Cu(II) and TEMPO redox partners exhibit cooperative reactivity and exploit the low-potential, proton-coupled TEMPO/TEMPOH redox process rather than the high-potential TEMPO/TEMPO+ process. The results show how electron-proton-transfer mediators, such as TEMPO, may be used in combination with first-row transition metals, such as copper, to achieve efficient two-electron electrochemical processes, thereby introducing a new concept for the development of non-precious-metal electrocatalysts.

Elucidation of the Transport Mechanism of Puerarin and Gastrodin and Their Interaction on the Absorption in a Caco-2 Cell Monolayer Model.

Puerarin (PUR) and gastrodin (GAS) are often used in combined way for treating diseases caused by microcirculation disorders. The current study aimed to investigate the absorption and transportation mechanism of PUR and GAS and their interaction via Caco-2 monolayer cell model. In this work, the concentration in Caco-2 cell of PUR and GAS was determined by HPLC method. The bidirectional transport of PUR and GAS and the inhibition of drug efflux including verapamil and cyclosporine on the transport of these two components were studied. The mutual influence between PUR and GAS, especially the effect of the latter on the former of the bidirectional transport were also investigated. The transport of 50 µg·mL-1 PUR in Caco-2 cells has no obvious directionality. While the transport of 100 and 200 µg·mL-1 PUR presents a strong directionality, and this directionality can be inhibited by verapamil and cyclosporine. When PUR and GAS were used in combination, GAS could increase the absorption of PUR while PUR had no obvious influence on GAS. Therefore, the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil.

Mechanistic study of the biosynthesis of R-phenylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations.

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cß atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4'-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4kcal mol-1 at 27 °C. From this point, the reaction continues with the abstraction of Hß atom of the HEThDP intermediate by the Oß atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9kcal mol-1 at 27 °C.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

The Infuence of Salicin on Rheological and Film-Forming Properties of Collagen.

Collagen films are widely used as adhesives in medicine and cosmetology. However, its properties require modification. In this work, the influence of salicin on the properties of collagen solution and films was studied. Collagen was extracted from silver carp skin. The rheological properties of collagen solutions with and without salicin were characterized by steady shear tests. Thin collagen films were prepared by solvent evaporation. The structure of films was researched using infrared spectroscopy. The surface properties of films were investigated using Atomic Force Microscopy (AFM). Mechanical properties were measured as well. It was found that the addition of salicin modified the roughness of collagen films and their mechanical and rheological properties. The above-mentioned parameters are very important in potential applications of collagen films containing salicin.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

N-Chlorosuccinimide (NCS)-N,N-dimethylformamide (DMF), a reagent for the oxidation of benzylic alcohols to aldehydes and ketones.

The oxidation of benzylic alcohol to corresponding aldehyde and ketone using N-chlorosuccinimide (NCS)-N,N-dimethylformamide (DMF) has been described. This method gives easy access to the corresponding carbonyl compounds under metal-free conditions, without the use of corrosive reagent at ambient temperature in good yield.

Ultrasound and Radiation-Induced Catalytic Oxidation of 1-Phenylethanol to Acetophenone with Iron-Containing Particulate Catalysts.

Iron-containing particulate catalysts of 0.1-1 µm size were prepared by wet and ball-milling procedures from common salts and characterized by FTIR, TGA, UV-Vis, PXRD, FEG-SEM, and XPS analyses. It was found that when the wet method was used, semi-spherical magnetic nanoparticles were formed, whereas the mechanochemical method resulted in the formation of nonmagnetic microscale needles and rectangles. Catalytic activity of the prepared materials in the oxidation of 1-phenylethanol to acetophenone was assessed under conventional heating, microwave (MW) irradiation, ultrasound (US), and oscillating magnetic field of high frequency (induction heating). In general, the catalysts obtained by wet methods exhibit lower activities, whereas the materials prepared by ball milling afford better acetophenone yields (up to 83%). A significant increase in yield (up to 4 times) was observed under the induction heating if compared to conventional heating. The study demonstrated that MW, US irradiations, and induction heating may have great potential as alternative ways to activate the catalytic system for alcohol oxidation. The possibility of the synthesized material to be magnetically recoverable has been also verified.

Special Issue: Development of Asymmetric Synthesis.

Biological systems usually respond differently to enantiomers of a chiral molecule due to the inherent chirality of the active receptor sites of enzymes in nature [...].

Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA+•), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetramethoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA+•, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA+• and that a different mechanism produces small ligninolytic oxidants during white rot.

Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations.

To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH-oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH-oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP+ . In small-scale reactions, the purified fusion enzymes show good performances, with 69-99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high-throughput oxidase-activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties.

Heterocyclic lactam derivatives containing piperonyl moiety as potential antifungal agents.

To study the novel functionalized heterocyclic molecules with highly potential biological activity, two series of heterocyclic lactam derivatives containing the piperonyl moiety were designed and synthesized. The newly obtained compounds have been identified on the basis of analytical spectral data, including 1H NMR, 13C NMR, and ESI-MS. The target compounds were evaluated for their potential antifungal activities in vitro against twelve species of the plant pathogen fungi (Sclerotinia sclerotiorum, Rhizoctonia solani, Rap Sclerotinia stemrot, Fusarium graminearum, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Alternaria tenuis Nees, Monilinia fructicola, Colletotrichum gloeosporioides, Phytophthora capsici, Magnaporthe oryzae). Preliminary bioassays suggested that all prepared compounds I1-14 displayed broad-spectrum and moderate antifungal activities compared with the positive control hymexazol, especially for Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, and Monilinia fructicola. In particular, the inhibition rate of compound I9 exhibited good inhibition activity reached 95.16% against Sclerotinia sclerotiorum, and compounds I5, I12 against Phytophthora capsici were 93.44%, 91.25%. Further studies revealed that compounds I5 (IC50 = 19.13 µM) and I12 (IC50 = 9.12 µM) exhibited obviously antifungal activities against Phytophthora capsici, which were better than that of commercial agricultural fungicide hymexazol (IC50 = 325.45 µM). Therefore, these target compounds could be further studied and explored as a lead skeleton for discovery of novel antifungal agents.

Reactive Polymorphic Nanoparticles: Preparation via Polymerization-Induced Self-Assembly and Postsynthesis Thiol-para-Fluoro Core Modification.

The use of 2,3,4,5,6-pentafluorobenzyl methacrylate (PFBMA) as a core-forming monomer in ethanolic reversible addition-fragmentation chain transfer dispersion polymerization formulations is presented. Poly[poly(ethylene glycol) methyl ether methacrylate] (pPEGMA) macromolecular chain transfer agents were chain-extended with PFBMA leading to nanoparticle formation via polymerization-induced self-assembly (PISA). pPEGMA-pPFBMA particles exhibited the full range of morphologies (spheres, worms, and vesicles), including pure and mixed phases. Worm phases formed gels that underwent a thermo-reversible degelation and morphological transition to spheres (or spheres and vesicles) upon heating. Postsynthesis, the pPFBMA cores were modified through thiol-para-fluoro substitution reactions in ethanol using 1,8-diazabicyclo[5.4.0]undec-7-ene as the base. For monothiols, conversions were 64% (1-octanethiol) and 94% (benzyl mercaptan). Spherical and worm-shaped nano-objects were core cross-linked using 1,8-octanedithiol, which prevented their dissociation in nonselective solvents. For a temperature-responsive worm sample, cross-linking additionally resulted in the loss of the temperature-triggered morphological transition. The use of the reactive monomer PFBMA in PISA formulations presents a simple method to prepare well-defined nano-objects similar to those produced with nonreactive monomers (e.g., benzyl methacrylate) and to retain morphologies independent of solvent and temperature.

Specificity of Herbivore Defense Responses in a Woody Plant, Black Poplar (Populus nigra).

The specificity of woody plant defense responses to different attacking herbivores is poorly known. We investigated the responses of black poplar (Populus nigra) to leaf feeding by three lepidopteran species (Lymantria dispar, Laothoe populi and Amata mogadorensis) and two leaf beetle species (Phratora vulgatissima and Chrysomela populi). Of the direct defenses monitored, increases in trypsin protease inhibitor activity and the salicinoid salicin were triggered by herbivore damage, but this was not herbivore-specific. Moreover, the majority of leaf salicinoid content was present constitutively and not induced by herbivory. On the other hand, volatile emission profiles did vary among herbivore species, especially between coleopterans and lepidopterans. Monoterpenes and sesquiterpenes were induced in damaged and adjacent undamaged leaves, while the emission of green leaf volatiles, aromatic and nitrogen-containing compounds (known to attract herbivore enemies) was restricted to damaged leaves. In conclusion, indirect defenses appear to show more specific responses to attacking herbivores than direct defenses in this woody plant.

United States Pharmacopeia Safety Review of Willow Bark.

Willow bark (Salix spp.) is an ingredient in some dietary supplements. No serious adverse effects were reported from trials of willow bark extracts delivering 120 - 240 mg salicin (the purported active constituent) daily for up to 8 weeks. All studies involved adults only; none involved special subpopulations such as pregnant or breastfeeding women, or children. The most common adverse effects associated with willow bark are gastrointestinal; a few allergic reactions were also reported. Some publications advise caution when taking willow bark. There is a risk of increased bleeding in vulnerable individuals, salicylates cross the placenta and are eliminated slowly in newborns, some persons are sensitive or allergic to aspirin, and children are at risk of Reye syndrome. Concurrent use with other salicylate-containing medicines increases these risks. Metabolism of 240 mg salicin from willow bark could yield 113 mg of salicylic acid, yet dietary supplement products are not required to be labeled with warnings. In contrast, over-the-counter low-dose aspirin (81 mg strength), which delivers 62 mg salicylic acid, is required by law to include cautions, warnings, and contraindications related to its use in pregnant and nursing women, children, and other vulnerable subpopulations, e.g., those using anticoagulants. In the interest of protecting public health, the United States Pharmacopeia has included a cautionary labeling statement in the United States Pharmacopeia Salix Species monograph as follows: "Dosage forms prepared with this article should bear the following statement: 'Not for use in children, women who are pregnant or nursing, or by persons with known sensitivity to aspirin.'".

Gentisyl Alcohol Inhibits Proliferation and Induces Apoptosis via Mitochondrial Dysfunction and Regulation of MAPK and PI3K/AKT Pathways in Epithelial Ovarian Cancer Cells.

Ovarian cancer is one of the prevalent gynecological cancers occurring in women. In particular, the efficiency of standard therapeutic methods decreases when recurrence and chemoresistance ensue. To assist standard anti-cancer agents in the cure of ovarian cancer, development and application of new compounds such as small molecules or natural products are required. Gentisyl alcohol is one of the secondary metabolites that can be obtained by purification from bacteria or fungi and is known to have antibacterial, antifungal, antiviral, and anti-cancer effects. In the present study, we verified the effect of gentisyl alcohol derived from marine Arthrinium sp. on suppressing proliferation and inducing apoptosis via DNA fragmentation in human ovarian cancers cells (ES2 and OV90 cells). We also confirmed that there was an accumulation of sub-G1 cells and a loss of mitochondrial membrane potential with calcium dysregulation in gentisyl alcohol-treated ovarian cancer cells. Moreover, gentisyl alcohol up-regulated signal transduction of MAPK and PI3K/AKT pathways. Collectively, our results demonstrated the possibility of gentisyl alcohol as a novel therapeutic agent for human ovarian cancer.

Kinetic analysis and degradation mechanism for natural attenuation of xylenes under simulated marine conditions.

Microcosm experiments were conducted to examine the attenuation of selected chemicals, i.e. m-xylene (MX), o-xylene (OX) and p-xylene (PX), under simulated marine conditions. Natural attenuation and the contribution of oxidation, photodegradation, biodegradation and volatilization to total attenuation were evaluated. The development of attenuation was in agreement with pseudo-first-order kinetics for all xylenes. The half-lives of MX, OX, and PX under optimal conditions were 0.76, 0.74 and 0.88 days, respectively. Attenuation kinetics were proposed to analyze the natural attenuation of xylenes. The leading attenuation type of MX, OX, and PX was volatilization, and the attenuation rate constants (KV) were 0.5587, 0.6733, and 0.4821 d-1, respectively. Biodegradation of OX (Kb: 0.0003 d-1) was extremely inhibited. The attenuation kinetics presented the attenuation of xylenes in microcosm. The reaction kinetics could be applied to analyze the natural attenuation of chemicals. MX and OX can be converted to one another under certain conditions. Toluene and ethylbenzene were detected for OX in the OP (oxidation and photodegradation) experiment under simulated marine conditions. 4-Methylbenzyl alcohol, p-methyl benzaldehyde and p-toluic acid, as the major intermediates, were identified during the natural attenuation of PX using GC/MS.