Evaluation Of Inhibitory Activity Research Articles

Evaluation of the alpha-amylase inhibitory activity of Euclea natalensis extracts used in the treatment of diabetes mellitus: An experimental and in silico approach

Evaluation of the alpha-amylase inhibitory activity of Euclea natalensis extracts used in the treatment of diabetes mellitus: An experimental and in silico approach

Isolation, Characterization, Genome Annotation, and Evaluation of Hyaluronidase Inhibitory Activity in Secondary Metabolites of Brevibacillus sp. JNUCC 41: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

Brevibacillus sp. JNUCC 41, characterized as a plant-growth-promoting rhizobacterium (PGPR), actively participates in lipid metabolism and biocontrol based on gene analysis. This study aimed to investigate the crucial secondary metabolites in biological metabolism; fermentation, extraction, and isolation were performed, revealing that methyl indole-3-acetate showed the best hyaluronidase (HAase) inhibitory activity (IC50: 343.9 μM). Molecular docking results further revealed that the compound forms hydrogen bonds with the residues Tyr-75 and Tyr-247 of HAase (binding energy: -6.4 kcal/mol). Molecular dynamics (MD) simulations demonstrated that the compound predominantly binds to HAase via hydrogen bonding (MM-PBSA binding energy: -24.9 kcal/mol) and exhibits good stability. The residues Tyr-247 and Tyr-202, pivotal for binding in docking, were also confirmed via MD simulations. This study suggests that methyl indole-3-acetate holds potential applications in anti-inflammatory and anti-aging treatments.

Abstract 1963: BB102, a highly selective and potent reversible-covalent FGFR4 inhibitor effective in multiple xenograft tumor models

Abstract Introduction: Fibroblast growth factor receptor 4 (FGFR4) belongs to the FGFR family which interact with fibroblast growth factors (FGFs) to regulate cell proliferation, differentiation and apoptosis. Due to unique structural domain, FGFR4 is specifically bound by FGF19 with high affinity in the presence of co-receptor β-Klotho. Hyperactivated FGF19/FGFR4 signaling, induced by FGF19/FGFR4 overexpression and FGFR4 mutation, was reported to be associated with the growth, metastasis and invasion of various cancer. Thus, FGFR4 is considered as a novel target to treat cancer with hyperactivated FGF19/FGFR4 signaling. Here we report BB102 as a highly selective and potent reversible-covalent FGFR4 inhibitor. Methods: BB102 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical and cell assays were applied in evaluation of BB102 inhibitory activity. Preclinical studies including pharmacokinetic (PK) and toxicity were performed in rats and dogs. Xenograft tumor models in mice were conducted to demonstrate the in vivo efficacy of BB102. Results: As a reversible-covalent FGFR4 inhibitor, about half compound of BB102 dissociated from FGFR4 kinase within 24 h, which was conducive to overcome the fast re-synthesis rate of FGFR4 (< 2 h) in hepatocellular carcinoma cells. BB102 only inhibited kinase activity of FGFR4WT, FGFR4N535K and FGFR4V550L at nanomolar levels in 207 kinase panel screening. BB102 completely blocked the phosphorylation of the downstream protein ERK at 16.7 nM. BB102 exhibited strong inhibitory effects on the cell proliferation in vitro and tumor growth in mouse models inoculated with the high expression of both FGF19 and FGFR4, or FGFR4 mutated tumor cells. PK profile of BB102 showed good oral bioavailability (F>70%) with a nearly linear dose-dependent exposure. The compound had no effects on the respiratory and central nervous systems in rats, cardiovascular system in Beagle dogs or hERG liability in vitro. In comprehensive safety evaluation, the Severely Toxic Dose in 10% (STD10) in rats was 300 mg/kg/day and the Highest Non-Severely Toxic Dose (HNSTD) in dogs were 100 mg/kg/day. Conclusion: BB102 is a highly selective and potent reversible-covalent FGFR4 inhibitor with promising efficacy in high expression of both FGF19 and FGFR4, and/or FGFR4 mutated tumor models and good safety in preclinical studies. The phase I study of BB102 is in progress. Citation Format: Qi Wang, Gongping Duan, Wei Hua, Xianglong Wei, Min Li, Junheng Wang, Xingmin Zhang. BB102, a highly selective and potent reversible-covalent FGFR4 inhibitor effective in multiple xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1963.

Synergizing structure and function: Cinnamoyl hydroxamic acids as potent urease inhibitors

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 µM. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

α-Glucosidase Inhibitory Phytochemical Components of Chinese Endemic Plant Whitfordiodendron filipes var. tomentosum.

Whitfordiodendron filipes var. tomentosum is an endemic plant in China. There have been no chemical or pharmacological studies of this plant reported before. In the current research, eight triterpenes and two steroids were obtained. Their structures were established by the analysis of NMR data and comparison with those reported in the literature. These ten structurally diverse compounds comprised five distinct carbon frameworks with different functionalities. The chemotaxonomic significance of these secondary metabolites was discussed, disclosing the common components between the variant W. filipes var. tomentosum and the species W. filipe. Evaluation of α-glucosidase inhibitory activities of these isolates disclosed that compounds 1, 2, 4, and 6 exhibited significant α-glucosidase inhibitory activities (IC50 = 16.6-19.2 μM), which were close in value to the positive control acarbose (IC50 = 11.5 μM). Moreover, the binding modes between the biologically active compounds 1, 2, 4, and 6 and the α-glucosidase protein were preliminarily studied using molecular docking. This study not only showed the chemical and biological profile of the plant W. filipes var. tomentosum but also revealed that these components could be developed as hypoglycemic lead compounds.

Identification and α-glucosidase inhibitory activity evaluation of two new coumarins derived from Mansonia gagei J. R. Drumm.

Five coumarins were isolated from the heartwood of Mansonia gagei, which included two newly discovered compounds, namely 11-hydroxypopulene E (1) and mansorin D (2), along with three previously identified compounds. The structures were determined through the utilisation of comprehensive spectroscopic data, ECD calculations, and a thorough comparison with existing literature data. The α-glucosidase inhibitory activities of all isolated compounds were assessed in yeast. Out of the compounds tested, compound 2 exhibited the most significant activity, displaying a percentage inhibition of 34.33% at a concentration of 200 μM.

Combinatorial preparation and structural characterization of anthocyanins and aglycones from Purple-heart Radish for evaluation of physicochemical stability and pancreatic lipase inhibitory activity

In this study, an efficient approach to preparation of different anthocyanins from Purple-heart Radish was developed by combining microwave-assisted extraction (MAE), macroporous resin purification (MRP) and ultrasound-assisted acid hydrolysis (UAAH) for evaluation of physicochemical stability and pancreatic lipase (PL) inhibitory activity. By optimization of MAE, MRP and UAAH processes, the anthocyanins reached the yield of 6.081 ± 0.106 mg/g, the purity of 78.54 ± 0.62 % (w/w) and the content of 76.29 ± 1.31 % (w/w), respectively. With high-resolution UHPLC-Q-Orbitrap/MS, 15 anthocyanins were identified as pelargonins with diverse glucosides and confirmed by pelargonidin standard. By glycosylation, pelargonins exhibited higher stability in different pH, temperature, light, metal ions environments than that of pelargonidin. However, PL inhibitory assay, kinetic analysis and molecular docking demonstrated that pelargonidin had higher PL inhibitory activity than pelargonins even though with similar binding sites and a dose–effect relationship. The above results revealed that the effect of glycosylation and deglycosylation on PL inhibitory activity and physicochemical stability.

Isolation, Characterization, Genome Annotation, and Evaluation of Tyrosinase Inhibitory Activity in Secondary Metabolites of Paenibacillus sp. JNUCC32: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

A potential strain, Paenibacillus sp. JNUCC32, was isolated and subjected to whole-genome sequencing. Genome functional annotation revealed its active metabolic capabilities. This study aimed to investigate the pivotal secondary metabolites in the biological system. Fermentation and extraction were performed, resulting in the isolation of seven known compounds: tryptophol (1), 3-(4-hydroxyphenyl)propionic acid (2), ferulic acid (3), maculosin (4), brevianamide F (5), indole-3-acetic acid (6), and butyric acid (7). Tryptophol exhibited favorable pharmacokinetic properties and demonstrated certain tyrosinase inhibitory activity (IC50 = 999 μM). For further analysis of its inhibition mechanism through molecular docking and molecular dynamics (MD) simulation, tryptophol formed three hydrogen bonds and a pro-Michaelis complex with tyrosinase (binding energy = -5.3 kcal/mol). The MD simulation indicated favorable stability for the tryptophol-mushroom tyrosinase complex, primarily governed by hydrogen bond interactions. The crucial residues VAL-283 and HIS-263 in the docking were also validated. This study suggests tryptophol as a potential candidate for antibrowning agents and dermatological research.

α-Glucosidase immobilization on calcium-sodium alginate-chitosan gel beads for inhibitory activity evaluation from traditional Chinese medicines

Discovering α-glucosidase inhibitors is one of the effective ways to develop new anti-diabetic drugs. In this study yeast α-glucosidase immobilized in calcium alginate-chitosan gel treated with polyethyleneimine and capillary electrophoresis analysis was used to select enzyme inhibitor compounds in 12 traditional Chinese medicine. The immobilized α-glucosidase shows enhanced tolerance to temperature (70 °C), excellent repeatability (RSD = 1.9%, n = 5), high stability and good reusability (retained 57.8% and 74% of its initial activity after storing at 4 ℃ for 30 days and reusing 10 times, respectively). Moreover, the Michaelis constant of immobilized α-glucosidase and half-maximal inhibitory concentration of acarbose were calculated to be 1.79 mM and 0.70 μM, respectively. These results demonstrate that the immobilized α-glucosidase for inhibitory activity evaluation is feasible. The immobilized α-glucosidase was applied to screen inhibitors from 12 traditional Chinese medicines, and Sanguisorbae Radix the root of Sanguisorba officinalis L. exhibited the strongest inhibitory activity on α-glucosidase.

In Vitro Evaluation of Alpha Amylase and Alpha Glucosidase Inhibitory Activity of Kamadhenu Ark extracts of Climate Smart Crop Millets

Diabetes Mellitus is characterized by abnormal Metabolism of glucose. The maintenance of glucose levels in blood without any side effects is still challenging in the medication of this disease. Various medical approaches like Allopathy, Homeopathy, Ayurveda, Unani etc are striving a lot for success in this field of medication. Present study is aimed to evaluate the Antidiabetic activity of Millets in combination with Kamadhenu Ark as solvent for the probable alpha Amylase and alpha Glucosidase inhibitory activities to reduce hyperglycemia. The Millets namely Paspalum scrobiculatum, Panicum miliaceum, Panicum sumatrense were selected for the study. The Kamadhenu Ark Extracts were prepared by Maceration method. The extracts were used for the estimation of alpha amylase and alpha-glucosidase inhibition activity. Extract of Panicum miliaceum exhibited highest α amylase and α glucosidase inhibitory potential with IC50 values of 0.239 and 1.526 mg/ml, respectively, which was comparable with acarbose (0.258 and 1.985 mg/ml). Extracts of Paspalum scrobiculatum and Panicum sumatrense have shown low activity. Present study paves a new path revealing the effective inhibition of α amylase and α glucosidase by Kamadhenu Ark extract of Panicum miliaceum (Proso Millet KPM).

Alpha-amylase inhibitory activity and in silico studies of in vitro sweet basil plantlets treated with chitosan and ZnO NPs

AbstractIn this study, an efficient protocol was developed to obtain multiplicated, healthy sweet basil plantlets with larger leaves and a greener color by culturing them on MS medium containing 1.0 mg/l 2iP. Chitosan and zinc oxide nanoparticles (ZnO NPs) were used as abiotic elicitors to maximize the main secondary metabolites (phenolic and flavonoid compounds and essential oil) in in vitro plantlets of sweet basil. Chitosan with a concentration of 4.0 mg/l recorded the maximum value of caffeic acid (1.189 mg/g DW), and 10 mg/l of ZnO NPs recorded the maximum content of rutin (0.936 mg/g DW). The plantlets cultured with 2.0 mg/l chitosan recorded the maximum percentage of essential oil (90.63%). 8,9-Dihydro-9-formyl cycloisolongifolene was the major compound (72.44%) and was detected in the plantlets treated with 2.0 mg/l chitosan. The inhibitory activity evaluation of α-amylase revealed that the maximum inhibitory activity was found in the plantlet control and those treated with 4.0 mg/l chitosan of the phenolic and flavonoid fractions. The maximum IC50 (7.36 ± 0.85) was found in the plantlet extract treated with 4.0 mg/l chitosan, followed by the plantlet control, which recorded 11.53 ± 0.86. Using AutoDuck 4.2 and PyMol software, it was revealed that rutin and caffeic acid compounds (the main active components) own hydrogen binding interactions and hydrophobic binding interactions with the active site of alpha-amylase from the porcine pancreas (PPA) enzyme with good binding energy (5.74 and 5.71 kcal/mol, respectively).

Evaluation of inhibitory activity, purification and X-ray crystallography of Alpha-Amylase inhibitor from Phaseolus vulgaris cultivars of Uttarakhand, India.

The present work is based on analysis of inhibitory activity of alpha-amylase inhibitor in selected cultivars of Phaseolus vulgaris of Uttarakhand. Fifteen samples were assessed for inhibitory activity of alpha-amylase inhibitor. Significant variations were found in different cultivars. Crude extract of alpha-amylase inhibitor from sample PUR (Purola) have shown maximum inhibitory activity (70.2 ± 0.84). Crude extract of all the cultivars have shown considerable variations in inhibitory activity in the temperature ranging from 20ºC to 100ºC. Based on inhibitory activity and heat stability profile, the alpha amylase inhibitor was purified from PUR cultivar. The purified inhibitor was found to be stable even at 90ºC with an inhibitory activity of 97.20 ±0.09. The molecular weight of purified inhibitor on Native PAGE (Polyacrylamide gel electrophoresis) was found to be 31kd, consisting of two subunits of 17kd and 14kd on SDS-PAGE.

The most commonly used spices in Thai traditional medicine: in vitro evaluation of anti-hyperglycemic, antioxidant, polyphenol content, and nitric oxide production inhibitory activities

Background and purpose: Diabetes mellitus is a persistent hyperglycemic condition. Thai cuisine and medicine incorporate spices: nutmeg, mace, clove buds, cardamom, cinnamon, and coriander. The in vitro impacts of these spices on anti-diabetic, antioxidant, anti-inflammatory, and total phenolic and flavonoid content were assessed. Experimental approach: Alpha-amylase and alpha-glucosidase inhibition assays were conducted. Antioxidant potential was measured through DPPH and ABTS assays. Anti-inflammatory activity was determined by inhibiting nitric oxide generation in RAW 264.7 cells. Total phenolic content was quantified using the Folin Ciocalteu method, while total flavonoid content was estimated via the aluminum chloride colorimetric method. Findings/Results: Ethanolic and aqueous extracts of a blend of spices (Siam cardamom, nutmeg, mace, and clove buds), denoted as 4-GlurE and 4-GlurA, displayed concentration-dependent inhibition of alpha-glucosidase, with IC50 values of 0.373 and 0.435 mg/mL, respectively. 4-GlurE and 4-GlurA exhibited antioxidant activity, by ABTS·+ radical and DPPH scavenging capabilities. 4-GlurE demonstrated anti-inflammatory potential by reducing nitric oxide generation (IC50: 43.95 ± 2.47 μg/mL). 4-GlurE and 4-GlurA possessed total phenolic content (TPC) of 122.47 ± 1.12 and 148.72 ± 0.14 mg GAE/g, respectively. 4-GlurE exhibited a higher total flavonoid content (TFC) compared to the aqueous extract (340.33 ± 4.77 and 94.17 ± 3.36 mg QE/g). Cinnamon and clove aqueous extracts were more potent than acarbose in alpha-glucosidase inhibition with the highest antioxidant activity. Polyphenol levels (TPC and TFC) exhibited strong correlations with antioxidant capacity. Conclusions and implications: Findings are consistent with the traditional use of 4-Glur, with cinnamon, for diabetes prevention and treatment.

Garcinia dulcis and Garcinia forbesii King fruit peel extract: Secondary metabolite composition, antioxidant, and elastase inhibitory activity evaluation.

Garcinia dulcis and Garcinia forbesii King are native plants from Indonesia and have tremendous potential as a source of raw medicines based on local wisdom. However, scientific data for strengthening pharmaceuticals are still limited. Therefore, it is necessary to conduct a study to strengthen and develop the potential of both plants using the approach of traditional medicine. This study aimed to explore the secondary metabolite composition and biological activity (antioxidant and antielastase) of both plants. Both samples were extracted using 70% ethanol and microwave-assisted extraction with a microwave power of 120 watts for 15 min. The extract obtained was then screened for phytochemicals using specific reagents. The total phenolic content (TPC) was determined using spectrophotometry with a 96-well microplate reader method. The total flavonoid content (TFC) was determined using the colorimetric method, whereas metabolite profiling analysis was conducted using the UPLC-QToF-MS/MS system. Meanwhile, biological activity was tested for antioxidant activity and antielastase as measured by a microplate reader 96-well spectrophotometry method at specific wavelengths. According to the results, G. dulcis and G. forbesii fruit peel extracts showed positive detection of particular secondary metabolites. TPC and TFC values were 13.98 ± 1.90 mg GAE/g and 10.33 ± 1.90 mg QE/g for G. dulcis and 11.98 ± 2.04 mgGAE/g and 1.96 ± 0.36 mgQE/g for G. forbesii. Metabolite profiling detected some compounds from G. dulcis, including ephedrannin B, hinokiflavone, mahuannin J, and candidate mass C9H12O8, and G. forbesii, including 5-Hydroxy-7,8,2'- trimethoxyflavone, lucialdehyde B, candidate mass C21H39NO4, candidate mass C14H10O6, and candidate mass C14H12O6. Meanwhile, the biological activities (antioxidant and antielastase) were 137.721 μg/mL and 108.893 μg/mL for G. dulcis and 481.948 μg/mL and 250.611 μg/mL for G. forbesii, respectively. Both plants showed different profiles of secondary metabolites and biological activities (antioxidant and antielastase) according to their respective characteristics.

Phytochemical Investigation and Biofilm-Inhibitory Activity of Bachtiari Savory (Satureja bachtiarica Bunge) Aerial Parts.

Satureja bachtiarica is an endemic plant from the Lamiaceae family, growing in the Zagros mountain range in Iran. Even if S. bachtiarica is reported to possess many biological activities, little is known about its chemical composition. For this reason, in the present research, a phytochemical investigation of this species was carried out. To have a preliminary metabolite profile of S. bachtiarica, the n-BuOH extract was analyzed using LC-ESI/LTQOrbitrap/MS/MS in negative ion mode, allowing the identification of specialized metabolites belonging to flavonoid, monoterpene, indol, phenylpropanoid, phenolic, lignan, coumarin, biphenyl, and triterpene classes. The LC-MS/MS analysis guided the isolation of compounds, and their structures were characterized using spectroscopic methods including 1D- and 2D-NMR experiments and HRMSn analysis. In this way, a compound never reported before belonging to the biphenyl class was identified. Total flavonoid content of the extract along with the antioxidant activity were assessed. Based on the traditional uses of S. bachtiarica suggesting potential antibacterial properties, an evaluation of the biofilm inhibitory activity of the extract and isolated compounds against mature biofilms of Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, as well as their influence on the metabolism of sessile bacterial cells, was conducted. The results evidenced that some compounds including parmentin B, biphenyls, and 1-(1H-indole-3-carboxylate)-β-D-glucopyranoside might inhibit some changes occurring in the bacterial cells, which increases their virulence. In particular, biphenyl derivatives at a concentration of 80 μg/mL were capable of limiting remarkably the mature biofilms of A. baumannii and L. monocytogenes remarkably at a percentage ranging between 52.76% and 75.02%, and they reached an inhibition percentage of 69.28 % against E. coli. Biphenyl derivatives were also effective in exerting an inhibitory action against the mature biofilm of P. aeruginosa (inhibition ranging from 59.38% to 81.08%) and Staphylococcus aureus (inhibition percentage reached 82.94%). Of note, the biphenyl derivatives resulted in being capable of acting on the metabolism of the cells within the biofilm of all five pathogens.

Novel acetylcholinesterase inhibitors: Synthesis, docking and inhibitory activity evaluation of 4-benzamido-N-(1-benzylpiperidin-4-yl) benzamide derivatives

The cholinergic hypothesis is one of the basic approaches for designing and discovering novel anti-Alzheimer drugs. Application of the pharmacophore of well-known drugs like donepezil helps us achieve new molecules. In the current project, a new series of benzamide derivatives (6a–6l) were designed and synthesized. Spectroscopic techniques (NMR, IR, MS) were utilized for characterization. Subsequently, Ellman’s protocol was carried out for acetylcholinesterase assay and the obtained results were compared to donepezil (IC50 = 0.6 ± 0.05 µM). Compound 6b which bears a chlorine atom at position meta of the phenyl ring was the most potent derivative in this series (IC50 = 0.14 ± 0.03 nM) and exhibited higher activity than donepezil. In addition, molecular docking was performed to explore the binding mode and related interactions. The in silico results demonstrated that compound 6b binds to the active site of the AChE through a hydrogen bond with Trp279. This compound could be suggested as a potential lead compound and more experimental and in vivo tests are needed to prove its eligibility as a drug candidate.

Design, Molecular Docking, Synthesis and Evaluation of Xanthoxylin Hybrids as Dual Inhibitors of IL‐6 and Acetylcholinesterase for Alzheimer’s Disease

AbstractBackgroundIL‐6 and AChE play a pivotal role in progression of AD, it is hypothesised that simultaneous inhibition of IL‐6 and AChE may prove as an effective strategy for treatment of AD. Therefore, the present study is undertaken to design novel molecules capable of acting as dual inhibitors of IL‐6 and AChE. Literature review reveals that xanthoxylin and a disubstituted amines or a carbamoyl amines are pharmacophores for IL‐6 and AChE inhibition, respectively.MethodXanthoxylin is coupled with various disubstituted amines or carbamoyl amines through alkyl linkers of different lengths (1‐4 carbon atoms) to design two series of 80 compounds. All designed compounds were docked into AChE to identify the best fit compounds for synthesis and evaluation of AChE inhibitory activity. The compounds showing >45% inhibition of EeAChE are selected for evaluation of IL‐6 and butyrylcholinesterase (BuChE) inhibitory activities. The compound found to be most potent against AChE, BuChE and IL‐6 inhibition was finally evaluated in vivo using STZ‐induced amnesia model in mice at three doses (0.2, 0.4 and 0.8 mg/kg).ResultCompound K13g is found to be the most potent inhibitor of EeAChE, BuChE and IL‐6. It is further evaluated in vivo, wherein it shows dose‐dependent effects. It is observed to be less active at lower doses, but completely reversed the STZ‐induced memory deficit at 0.8 mg/kg.ConclusionThe findings suggest that a compound having a xanthoxylin moiety connected to a piperazine ring through a three‐carbon atom chain may provide a useful template for the development of new chemical entities effective against AD.

Evaluation of Cytotoxic, COX Inhibitory, and Antimicrobial Activities of Novel Isoxazole-carboxamide Derivatives

Abstract: Isoxazole derivatives are one of the heterocyclic structures that have various biological activities. Objective: This study aimed to design and synthesize novel isoxazole derivatives and evaluate their cytotoxic, cyclooxygenase (COX) inhibitory, and antimicrobial activities. Methods: Coupling reactions of aniline derivatives and isoxazole carboxylic acid have been established to synthesize chloro-fluorophenyl-isoxazole carboxamide derivatives. The synthesized compounds were characterized using 1H, 13C-NMR, IR, and HRMS spectrum analysis and evaluated by MTS, COX kit, and antimicrobial microdilution assays. Results: The synthesized compounds showed moderate to potent cytotoxic activity against all the screened cancer cell lines (except 2b against HepG2) with an IC50 range of 0.107-77.83 μg/ml. The results showed that the most potent compound against cervical cancer cell line (HeLa) was the 2b compound, with an IC50 value of 0.11±0.10 μg/ml, which is less than the IC50 for the potent anticancer drug Doxorubicin. While the 2a and 2b compounds have potential antiproliferative activities against Hep3B with IC50 doses of 2.774±0.53 and 3.621±1.56 μg/ml, respectively. Furthermore, 2c compound was the most active against MCF7, with an IC50 value of 1.59±1.60 μg/ml. In addition, the most potent isoxazole derivative against the COX1 enzyme was the 2b compound, with an IC50 value of 0.391 μg/ml, and compound 2a had a good selectivity ratio of 1.44 compared to the Ketoprofen positive control. However, compound 2c showed antifungal activity against Candida albicans with an MIC value of 2.0 mg/ml in comparison to the antifungal drug Fluconazole (MIC = 1.65 mg/ml). Conclusion: The synthesized compounds could be candidates for anticancer drugs in the future, and other analogues and cytotoxicity evaluations should be conducted.

Scots pine defensins inhibit Ips acuminatus α-amylase activity

Background. Pine bark beetle Ips acuminatus (Gyllenhal, 1827) is one of the most harmful pests of pine trees as it affects the phloem of the upper part of the stem and branches, disrupting the flow of nutrients and water to the crown. I. acuminatus feeds by plant tissues rich in starch, so α-amylases must play a pivotal role in the carbohydrate metabolism of these insects. However, in conifer bark beetles, α-amylases remain poorly understood. Materials and Methods. To detect the α-amylase activity in the digestive system of I. acuminatus, we obtained extracts from larvae, pupae, and adults that were collected from naturally infested Scots pine. The α-amylase activity of crude extracts from different stages and parts of the bark beetle’s body was assessed using 1% starch agar plates. The quantitative evaluation of the α-amylase inhibitory activity of recombinant defensins PsDef1, PsDef2, and PsDef5.1 was performed using the Bernfeld method. The docking models of Scots pine defensins and Ips typographus L. α-amylase (AmyIp) complexes were predicted using the ClusPro 2.0 web server. Results and Discussion. As a result, we found the presence of α-amylase activity in the digestive systems of both larvae and adults of I. acuminatus, but not in pupae. All tested defensins, PsDef1, PsDef2, and PsDef5.1, exhibited inhibitory activity against insect α-amylase at micromolar concentrations. The IC50 values for these peptides were 4.9±0.6 μM, 4.6±0.8 μM, and 2.8±0.5 μM, respectively. In the PsDefs-AmyIt complexes, a network of hydrogen bonds, ionic bridges, and nonbonded contacts are formed between the enzyme and its inhibitor, which prevents the substrate from reaching the catalytic site. The PsDef5.1-AmyIt complex has the largest interfacial contact area, 2328 Å2, in comparison with two other defensins, which correlates well with the inhibitory activity of defensins in this study. Conclusion. Thus, we have identified α-amylase activity in I. acuminatus and demonstrated the ability of Scots pine defensins to inhibit it, sugges­ting that they play a role in pine defenses against this pest.

Mass spectrometry as a tool for the dereplication of specialised metabolites from Pterocaulon angustifolium DC

Pterocaulon genus comprises 26 species, half of them have been phytochemical investigations regarding the chemical composition, and coumarins have been considered the chemotaxonomic markers in the genus. Herein Pterocaulon angustifolium DC (Asteraceae), a native plant from Brazil, is investigated for the first time. Twenty-six compounds were isolated from aerial parts of P. angustifolium DC., being 5 triterpenes, 4 phytosterols, 9 flavonoids, 3 phenolic acids, and 5 coumarins. Moreover, a total of 177 compounds were putatively identified using the dereplication technique by UHPLC-HRMS/MS, more than 50% correspond to flavonoids and coumarins. Although 41 different coumarins have already been reported in Pterocaulon genus, 16 were identified for the first time in this study. Crude ethanolic extract and fractions of P. angustifolium were also biologically investigates, and dichloromethane fraction was the most active fraction in the evaluation of antiproliferative, antioxidant, antimicrobial and cholinesterase inhibitory activities.