STUDYING BIOLOGICAL ACTIVITIES OF GLUCOSAMINE SCHIFF BASES BASED ON APIS MELLIFERA

ABSTRACT

Glucosamine and its derivatives have attracted increased attention in biomedicine and related scientific fields in recent years due to their pronounced biological activity. Of particular interest are Schiff bases synthesized based on glucosamine, which demonstrate promising cytotoxic activity against tumor cells. In this study, we obtained new Schiff bases derived from glucosamine. The resulting compounds showed significant antimelanoma activity, as well as activity against colorectal cancer cells. Infrared (IR) spectroscopy methods were used for structural identification, and biological activity was evaluated through experimental biotests, prediction using the online PASS service, and molecular docking.

АННОТАЦИЯ

Глюкозамин и его производные в последние годы привлекают повышенное внимание в биомедицине и смежных научных направлениях благодаря выраженной биологической активности. Особый интерес вызывают основания Шиффа, синтезированные на основе глюкозамина, которые демонстрируют перспективную цитотоксическую активность в отношении опухолевых клеток. В рамках настоящего исследования нами были получены новые основания Шиффа на основе глюкозамина. Полученные соединения продемонстрировали значительную антимеланомную активность, а также активность в отношении клеток колоректального рака. Для структурной идентификации были применены методы инфракрасной (ИК) спектроскопии, а для оценки биологической активности — экспериментальные биотесты, прогнозирование с использованием онлайн-сервиса PASS и молекулярный докинг.

Keywords: Apis mellifera, chitosan, glucosamine hydrochloride, Schiff bases, Pass online, Molecular Docking.

Ключевые слова: Apis mellifera, хитозан, гидрохлорид глюкозамина, основания Шиффа, Pass онлайн, молекулярный докинг.

Introduction

Glucosamine (2-amino-2-deoxy-D-glucose), also known as chitosamine (Scheme 1), is a water-soluble amino sugar belonging to the class of hexosamines. It is typically obtained through the hydrolysis of chitin or chitosan and is present in various human tissues. Unlike carbohydrates, which primarily serve as a source of energy, amino sugars are integral components of tissue structures. Glucosamine is a naturally occurring glycoprotein present in the connective tissues of the human body and the mucous membranes of the gastrointestinal tract [1]. This monosaccharide plays a crucial role in the biosynthesis of glucosaminoglycans and proteoglycans, which are essential components of various tissues, including nails, tendons, skin, eyes, bones, and heart valves, as well as the extracellular matrix of connective tissues. Additionally, glucosamine exhibits antioxidant and anti-inflammatory properties. It is primarily utilized for pain relief, managing inflammatory bowel disease, and as a dietary supplement available in forms such as glucosamine, glucosamine hydrochloride, or glucosamine sulfate [2,3].

Figure 1. Extraction of glucosamine hydrochloride by hydrolysis of chitosan

Recently, numerous studies have focused on the synthesis of Schiff bases, which demonstrate a wide range of biological activities [4–6], including antifungal, antibacterial, antimalarial, antiproliferative, anti-inflammatory, antiviral, antimicrobial, and antipyretic effects. It is particularly important to synthesize Schiff bases derived from D-glucosamine, given their significant biological properties. Glucosamine exhibits notable activity against cancer cells while maintaining low cytotoxicity toward normal human cells [7]. Additionally, literature evidence suggests that incorporating a sugar moiety into the structure of Schiff bases can reduce toxicity and enhance anticancer activity. Furthermore, the presence of hydroxyl groups in glucosamine increases water solubility, which is advantageous for biological applications [4]. The use of glucosamine as a primary precursor in the synthesis of Schiff bases is significant for applications in biomedicine and various other fields [8]. In our study, glucosamine hydrochloride was obtained through the hydrolysis of chitosan derived from local raw material, Apis mellifera [9]. Subsequently, Schiff bases of glucosamine hydrochloride were synthesized and their biological activities are studied using PASS online and Molecular Docking software.

Materials and methods

In this experiment, obtained compouds were characterized by IR spectroscopy (IRTracer-100, SHIMADZU, 2017; 4000-500 sm^-1, BRUKER Fourier spectrometer; 4000-400 sm^-1) and AutoDock 4.2 software.

Preparation of glucosamine hydrochloride via hydrolysis of chitosan

1.15 g of chitosan was weighed and dissolved in 15 ml of 30% hydrochloric acid (HCl). The mixture was then heated at 80 °C for 4 hours. After cooling, an additional 15 ml of distilled water was added, and the solution was filtered. A portion of the solvent present in the filtrate was evaporated to concentrate the solution. The resulting clear solution was refrigerated for 24 hours to facilitate the crystallization of glucosamine hydrochloride as white crystals. The precipitate was subsequently collected by filtration, washed with ethanol, and dried in an oven at 40 °C (0.23 g; η = 20%).

Synthesis of Schiff bases derived from glucosamine hydrochloride

First, 0.1 g of glucosamine hydrochloride was measured and dissolved in 2 mL of distilled water. Subsequently, 0.085 g of salicylic aldehyde was dissolved in 1.5 mL of ethanol. The two solutions were combined in a molar ratio of 1:1.5, resulting in an orange-colored solution. To this mixture, 0.02 g of NaOH was added. The reaction was then conducted under stirring on a magnetic stirrer at a temperature of 70–80°C for a duration of 3 hours. After the reaction was complete, the solution was stored in a refrigerator for 24 hours to facilitate complete precipitation of the formed substance. Consequently, yellow crystals were obtained. The precipitate was then filtered, washed with ethyl alcohol, and dried in an oven at 40 °C (0.124 g; η = 78%).

Additionally, Schiff bases of glucosamine hydrochloride with indole-3-aldehyde (yield 69%) and 6-nitro-1,3-dioxy-8-carbaldehyde (yield 74%) were synthesized using the aforementioned method.

Results and discussions

IR Spectra Studies

When comparing the IR spectra of glucosamine hydrochloride and chitosan, the spectrum of the hydrolysis product exhibits a prominent absorption band around 3283 cm⁻¹, corresponding to the N–H stretching vibration of the NH₂ group. Additionally, the intensities of the vibrational bands associated with the asymmetric and symmetric stretching vibrations of C–H bonds decrease and shift to approximately 2943 cm⁻¹ and 2884 cm⁻¹, respectively. The loss of vibrational frequencies associated with amide bonds, along with the emergence of intense vibrational bands resulting from the asymmetric deformation vibration of the NH₂ group at 1615 cm⁻¹ and symmetric deformation vibrations at 1583 and 1537 cm⁻¹, was observed (Fig. 1) [10]. Additionally, notable alterations in the vibrational frequencies of glycosidic bonds and pyranose rings suggest that hydrolysis has occurred.

Schiff bases of some aromatic aldehydes (salicylaldehyde, indole-3-aldehyde, 6-nitro-2,4-dihydro-1,3-benzodioxin-8-carbaldehyde) obtained during our research were synthesized.

When the IR spectra of the synthesized salicylaldehyde Schiff base and glucosamine are compared, notable changes are observed. Specifically, the disappearance of vibrational frequencies associated with the valence and deformation vibrations of the NH₂ group indicates its involvement in the reaction. Additionally, new, intense vibrational bands appear at 1627 cm⁻¹—corresponding to the C=N stretching vibration of the imine linkage—and at 1491, 1409, and 765 cm⁻¹, which are attributed to vibrational modes associated with the aromatic group attached to glucosamine. These emerging bands suggest the formation of a Schiff base (Fig. 2) [11]. Furthermore, the IR spectra of Schiff bases derived from indole-3-aldehyde and 6-nitro-2,4-dihydro-1,3-benzodioxin-8-carbaldehyde with glucosamine display characteristic C=N stretching vibrations at 1650 and 1688 cm⁻¹, respectively, further supporting Schiff base formation.

STUDYING BIOLOGICAL ACTIVITIES

PASS online results

The biological activities of Schiff bases derived from glucosamine were evaluated using the PASS online program. The computational results indicate that the synthesized Schiff bases exhibit higher predicted activity against colon cancer, melanoma, tuberculosis, mycobacteria, and as apoptosis agonists compared to original glucosamine. These findings suggest potential enhancements in biological activity conferred by the Schiff base modifications. Consequently, the results provide insights into the biological properties of both glucosamine and its Schiff base derivatives, with particular emphasis on several specific activities that have been extensively investigated. Among glucosamine (III) and synthesized Schiff bases, it was studied that glucosamine itself exhibits the highest antitumor activity of 0.825 (Pa), and also that Schiff base (IV) with salicylaldehyde 0.781 (Pa) and Schiff base (V) with indole-3-aldehyde 0.768 (Pa) also exhibit activity at values ​​close to glucosamine. It was found that salicylaldehyde Schiff base 0.360(Pa) and indole-3-aldehyde Schiff base 0.370(Pa) activity against colon cancer, salicylaldehyde Schiff base 0.40(Pa) and indole-3-aldehyde Schiff base 0.570(Pa) activity against melanoma tumor. Also, Schiff base with salicylaldehyde showed the highest activity against tuberculosis 0.449(Pa) and mycobacteria 0.553(Pa). As an agonist of apoptosis, 6-nitro-1,3-dioxy-8-carbaldehyde Schiff base (VI) 0.547 (Pa) showed the highest activity. The obtained results allow us to conclude about the biological activities of glucosamine and Schiff bases.

MOLECULAR DOCKING RESULTS

Molecular docking results of colon cancer protein

The 6ULM protein structure was retrieved from the Protein Data Bank (PDB) website. It was prepared by removing any additional compounds, adding hydrogen atoms, and assigning Kollman charges using AutoDock Tools. The ligands—glucosamine, indole-3-aldehyde, 6-nitro-1,3-dioxy-8-carbaldehyde, and salicylaldehyde Schiff bases — were modeled in Avogadro and saved in PDB format. Docking studies conducted with AutoDock 4.2 revealed that the binding energy between the indole-3-aldehyde Schiff base and the 6ULM protein was the lowest, indicating the strongest binding affinity among the tested ligands. The minimum energy value indicative of bond strength was ΔG_binding = -5.55 kcal/mol. The amino acid residues TYR17, SER15, GLN20, TYR58, PHE14, ILE26, ILE25, GLN24, SER23, and ILE16 participate in the formation of the covalent bond between indole-3-aldehyde Schiff base and the 6ULM protein. Additionally, ILE25, GLN24, and SER23 are involved in hydrogen bonding interactions between the protein and the ligand (Table 1).

Table 1.

Binding energies between ligand and protein (ΔG_binding, kkal/mol)

Molecular docking results for melanoma tumor protein

7UOA MTP-1, a protein belonging to the Melanoma Antigen A4 family, was retrieved from the Protein Data Bank (PDB) database. The structure was prepared by removing extraneous compounds, adding hydrogen atoms, and assigning Kollman charges using AutoDock Tools. Ligands—including glucosamine, indole-3-aldehyde, 6-nitro-1,3-dioxy-8-carbaldehyde, and salicylic aldehyde Schiff bases—were modeled in Avogadro and saved in PDB format. Molecular docking studies conducted with AutoDock 4.2 revealed that the indole-3-aldehyde Schiff base exhibited the lowest binding energy with the 7UOA MTP-1 protein, indicating a stronger binding affinity between this ligand and the protein. The lowest energy value indicative of bond strength was ΔG_binding = -5.61 kcal/mol. The amino acid residues GLN195, ARG126, LEU122, ILE196, ARG123, PHE197, HIS119, ASP115, and GLU116 participate in the formation of a bond between indole-3-aldehyde Schiff base and the 7UOA MTP-1 protein. Specifically, the residues ARG123, GLN195, HIS119, and ILE196 are directly involved in this interaction (Table 2).

Table 2

Binding energies between ligand and protein (ΔG_binding, kkal/mol)

Conclusion

Glucosamine hydrochloride, which exhibits high biological activities, was obtained by acidic hydrolyzing of chitosan extracted from Apis mellifera, which is a local raw material. Thereafter, Schiff bases of obtained glucosamine were synthesized and their structures were analyzed using the IR-spectroscopy method. Analysis of the biological activities of the synthesized Schiff bases using PASS online and molecular docking programs revealed that the Schiff base derived from indole-3-aldehyde exhibits significant activity against colon cancer and melanoma tumors.

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