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Антиоксидантные свойства флавоноидов мирицетина (М1), мирицитрина (М2) и мирицетин-3'-O-сульфата (М3), выделенных из корней Limonium caspium (Willd.) P.Fourn (Plumbaginaceae) из флоры Азербайджана были сравнительно изучены in vitro, а биологическая активность in silico (PASS Online, ProTox 3.0, SwissADME, SwissTarget и ADMETlab 3.0). Также изучены взаимоотношении лиганд – макромолекула с помощью программы молекулярного докинга. Методом DPPH рассчитана антиоксидантная активность IC50 веществ (мкг/мл): М1(7.81), М2(6.34) и М3(6.2). Предсказано, что М1 обладает (%) выраженными противоопухолевыми (92.4), а М2 (99.0) и М3 (99.1) кровоостанавливающими свойствами. Молекулярный докинг был выполнен с использованием программы AutoDock Vina 4.2. В качестве исследуемого лиганда использовалось соединение M3, а в качестве белка-мишени была выбрана ксантиндегидрогеназа. Молекула M3 связывается с активным сайтом фермента ксантиндегидрогеназы посредством нескольких различных механизмов взаимодействия и общий профиль взаимодействий свидетельствует о сильном и специфическом связывании M3 с ферментом.

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INTRODUCTION Plants of the genus Limonium are rich in biologically active phenolic compounds, and the species Limonium caspium (Willd.) P. Fourn. (Plumbaginaceae) has widely grown in the flora of Azerbaijan [1]. Antioxidants are compounds capable of neutralizing the harmful effects of free radicals and protecting cells from oxidative stress. Free radicals can damage cellular membranes, DNA, and proteins, thereby contributing to the development of cancer, cardiovascular diseases, neurodegenerative disorders, and other chronic pathologies. Phenolic compounds of plant origin, particularly flavonoids usually considered potent natural antioxidants. They exert their antioxidant effects both by scavenging free radicals through hydroxyl groups and by chelating metal ions. The antioxidant activity of flavonoids is commonly assessed in vitro using DPPH, ABTS, FRAP, and CUPRAC assays. Numerous studies have demonstrated that flavonoids isolated from L. caspium (Willd.) P. Fourn. exhibit pronounced antioxidant properties, highlighting their potential medical and pharmacological relevance [2].  In silico studies encompass the analysis of biological processes and molecular interactions through computer-based modeling and bioinformatics tools.The aim of the present study is to investigate the antioxidant properties of flavonoids in vitro, as well as to perform a comparative in silico assessment of their biological activity, physicochemical properties, solubility, bioavailability, and pharmacokinetic parameters, along with molecular docking analysis of myricetin (M1), myricitrin (M2), and myricetin-3′-O-sulfate (M3) isolated from L. caspium.

MATERIALS AND METHODS In vitro determination of antioxidant activity. The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed according to the method described by Blois et al. and adapted for a 96-well microplate format [3]. This method assesses the effect of antioxidant-active compounds on the DPPH radical solution. The unpaired electron of DPPH accepts a hydrogen atom from the tested compound, resulting in the formation of the corresponding hydrazine. The antioxidant activity index, IC50, represents the concentration of a compound required to inhibit 50% of DPPH radical absorbance at a wavelength of 517 nm; lower IC₅₀ values indicate stronger antioxidant activity. Compounds M1, M2, and M3 were initially dissolved in methanol, and serial dilutions at different concentrations (1–20 µg/mL) were prepared to determine optimized IC50 values. Gallic acid (Sigma Aldrich, batch SLCN 0435) was used as a positive control, dissolved in methanol and six analytical solutions were prepared within the concentration range of 0.5–5.0 µg/mL. A fresh DPPH solution at a concentration of 0.060 mg/mL was prepared daily by dissolving 6 mg of DPPH (Sigma Aldrich, D9132-5G, batch 0000187174) in 100 mL of methanol and allowing the solution to keep in the dark at room temperature for 1–3 hours before the analysis. The DPPH assay was carried out in a 96-well microplate. The first row served as a blank row and contained methanol only, while the second row represented the negative control containing methanol and the DPPH reagent. In the subsequent rows, analytical solutions of each sample were added in triplicate, along with an additional row containing only the sample solution without DPPH. The composition of each well was as follows: Blank wells: 250µL methanol (MeOH) Negative control wells: 50 µL MeOH + 200µL DPPH solution in MeOH Sample or positive control wells: 50µL sample or positive control + 200 µL DPPH solution. Sample blank wells: 50µL sample + 200 µL MeOH The 96-well microplate was covered with a lid and incubated in a spectrophotometer (Agilent BioTek Epoch, USA) for 1 hour. Absorbance measurements were recorded at 517 nm. Antioxidant activity and IC₅₀ values (defined, as the concentration required inhibiting 50% of DPPH radicals) were calculated using established equations [4].

In silico assessment of biological activity. For the comparative in silico assessment of the biological activity of flavonoids M1, M2, and M3, the PASS Online, SwissADME, SwissTargetPrediction, and ADMETlab 3.0 platforms were employed. Molecular docking simulations of ligand–macromolecule interactions were conducted using AutoDock 4.2. Compound M3 was selected as the ligand, while xanthine dehydrogenase was chosen as the target macromolecule. The crystal structures of the target proteins were retrieved from the Protein Data Bank (https://www.rcsb.org; PDB IDs: 1BML, 1JRO). The PDB format of the ligand (compound M3) was generated using MarvinSketch software (https://chemaxon.com/ marvin). Protein preparation: the downloaded crystal structure of xanthine dehydrogenase was prepared prior to molecular docking simulations. Initially, all non-essential molecules, including co-crystallized ligands, solvent molecules, and water residues, were removed using Discovery Studio Visualizer software (https://discover.3ds.com/discovery-studio-visualizer download). Subsequently, the cleaned protein structures were processed in AutoDock Tools version 1.4.5. During this step, polar hydrogen atoms were added to the protein structures, and Kollman partial charges were assigned to all atoms to ensure accurate representation of electrostatic interactions. The prepared protein structures were then saved in PDBQT format, which is required for molecular docking calculations, and were used as receptor models in subsequent docking studies. Grid box parameters were set to dimensions of 80 × 80 × 80 Å. Molecular docking simulations were performed using AutoDock 4.2 via the command-line interface [5].

RESULTS AND DISCUSSION. In vitro antioxidant activity The antioxidant activity of methanolic solutions of M1, M2, and M3 at various concentrations was assessed using the DPPH assay. The resulting dose–response curves are presented in Figures 1 and 2.According to the program predictions, compound M1 exhibits pronounced antitumor activity (92.4%), anticancer activity against prostate cancer (86.8%), antimutagenic activity (78.3%), as well as other biological activities.Investigation of M1, M2, and M3 using the SwissADME program Figure 3 presents the bioavailability radar diagrams for compounds M1, M2, and M3. The radar plots illustrate the main physicochemical parameters affecting oral bioavailability, including LIPO (lipophilicity), SIZE (molecular size), POLAR (polarity), INSOLU (solubility), INSATU (degree of saturation), and FLEX (molecular flexibility). Localization of the red polygon within the pink optimal area indicates a higher likelihood of drug-like properties. The distribution of these parameters differs among M1, M2, and M3, suggesting variations in their bioavailability potential. Overall, the radar diagrams provided by the SwissADME program allow a comparative assesment of the compounds from an ADME perspective.Physicochemical properties, water solubility, pharmacokinetics, lipophilicity, drug-likeness, and medicinal chemistry parameters were predicted using the SwissADME software (Tables 5–9). Thus, it was determined that the macromolecules with which M1 is most likely to interact are kinase (26.7%) and enzyme (20%). The macromolecules most likely to bind with M2 are lyase (26.7%), enzyme (26.7%), and G protein-coupled receptor family A (20%). Considering the high antioxidant activity and the rare occurrence of compound M3 compared to M1 and M2 (Table 1), special attention was given to this compound. The macromolecule with which M3 is most likely to bind and exhibit antioxidant properties was identified as xanthine dehydrogenase, belonging to the oxidoreductase class. Under pathological conditions such as ischemia, xanthine dehydrogenase converts into xanthine oxidase, which actively produces free radicals. It is hypothesized that M3, by binding to xanthine dehydrogenase in the body, prevents the conversion of the enzyme into xanthine oxidase. Comparative study of M1, M2, and M3 using ADMETlab 3.0. This program predicted several parameters that differ from those determined by SwissADME regarding physicochemical properties (Table 10). Molecular docking of compound M3 The compound M3 was used as the ligand, while xanthine dehydrogenase was selected as the macromolecular target. The 2D interaction structure is presented in Figure 5, and the 3D structures are shown in Figures 6 and 7. The binding energy of compound M3 upon interaction with the xanthine dehydrogenase enzyme is provided in Table 11.  Interaction of molecule M3 (ligand) with xanthine dehydrogenase enzyme in 2D format. The analysis revealed the following key interactions:
1.Van der Waals interactions: Molecule M3 forms weak Van der Waals contacts with several amino acid residues, primarily with Leu (A:216, B:688), Val (B:94, B:264), Ile (B:118), Ala (B:116), Gly (B:38), and Phe (B:99). These interactions contribute to the stable binding of the ligand within the enzyme's active site.                               
2. Hydrogen bonds:  Hydrogen bonds are formed between molecule M3 and the amino acid residues Arg (B:110, B:114). These bonds provide high selectivity for ligand binding at the active site and help stabilize the complex.                                 
3. Carbon-hydrogen bonds: The figure illustrates carbon-hydrogen bonds formed between the aromatic and hydroxyl groups of the ligand and the residues Val (B:264) and Thr (B:41).                                                                   
4. π-alkyl interactions: π-Alkyl interactions occur between the aromatic π-systems and alkyl groups with residues Ala (B:113) and Arg (B:114). These hydrophobic interactions enhance the ligand’s firm positioning in the active center.
5.Negative (unfavorable) interaction: It is important to note that a negative interaction is observed with residue Asp (B: 261), which may cause electrostatic repulsion of a part of the ligand molecule and potentially weaken the binding strength. Overall, molecule M3 binds to the active site of xanthine dehydrogenase through several different interaction mechanisms. Hydrogen bonds and π-alkyl interactions play a particularly significant role in complex stabilization. Van der Waals and carbon-hydrogen contacts provide additional stabilizing effects. Although the negative interaction may partially affect the binding energy, the overall interaction profile indicates strong and specific binding of M3 to the enzyme.

CONCLUSION In this study has been carried out the comparative in vitro investigation of the antioxidant properties, as well as in silico assessment of the biological activity, physicochemical properties, solubility, bioavailability, pharmacokinetic parameters, and drug-likeness of flavonoids M1, M2, and M3 isolated from Limonium caspium species growing along the Caspian Sea coast in Azerbaijan. In vitro antioxidant activity testing.The antioxidant activities of flavonoids M1, M2, and M3 were examined using the DPPH assay. As a result of the analysis, the IC50 antioxidant activity values were calculated, and the following results were obtained: M1 (7.81 μg/ml), M2 (6.34 μg/ml), and M3 (6.2 μg/ml), indicating that the substances possess a strong antioxidant effect. Gallic acid was used as a positive control with an IC₅₀ value of 3.48 µg/mL.

In silico biological activity study. Computational tools including PASS Online, SwissADME, SwissTarget, and ADMETlab 3.0 were applied to predict the biological activities of flavonoids M1, M2, and M3. According to PASS Online predictions, M1 exhibits significant antitumor activity (92.4%), anticancer activity against prostate cancer (86.8%), antimutagenic activity (78.3%), among other effects. M2 demonstrated high hemostatic (99.0%), cardioprotective (98.3%), vasoprotective (96.2%), and additional activities. M3 showed pronounced hemostatic activity (99.1%) along with other notable biological properties. Molecular docking was performed using AutoDock Vina 4.2, where compound M3 was used as the ligand and xanthine dehydrogenase was selected as the protein target. It is suggested that M3 may bind to xanthine dehydrogenase in the body, potentially preventing its conversion into xanthine oxidase.

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Ключевые слова

• Limonium caspium (Willd.) P.Fourn.
• мирицетин
• мирицитрин
• мирицетин-3՛-O-сульфат
• антиоксидантность
• in silico
• молекулярный докинг
• in vitro.

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Опубликовано: 14.May.2026

DOI: https://doi.org/10.28942/atuj.v6i2y2026.145

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