Characterization and investigation of cytotoxicity and antimicrobial properties of coencapsulated limonene and thymol into the Ferula assafoetida gum microparticles
Characterization and investigation of cytotoxicity and antimicrobial properties of coencapsulated limonene and thymol into the Ferula assafoetida gum microparticles
amirhossein tashakor,1,*davood mansouri,2atefe rezaei,3
1. Department of Microbiology, school of Medicine, Isfahan University of medical sciences, Iran 2. Department of Microbiology, school of Medicine, Isfahan University of medical sciences, Iran 3. Department of Food Science and Technology, School of Nutrition and Food Science, Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Introduction: The connection between antimicrobial resistance and a heightened risk of treatment failure and recurrence is well-established. The six hospital-acquired pathogens, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., are collectively referred to by the acronym ESKAPE are the main culprits behind hospital infections worldwide, with a majority being multidrug-resistant strains, thereby posing a significant challenge to clinical practice. Thymol (Th) is derived from cymene and is a naturally occurring phenol monoterpene. It is an isomer of carvacrol. D-limonene (L) is a vital element of citrus flavor obtained from the peel and pulp of citrus fruits. Both limonene and thymol exhibit low stability and are prone to oxidation when exposed to air, light, humidity, and high temperatures. In this study, encapsulation of Th and L in Assafoetida gum (AFG) was investigated.
Methods: The characterization of the obtained encapsulated complexes was done by scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analyzer (TGA). Also, Encapsulation efficiency, and antibacterial properties of the free L and Th and encapsulated ones measured. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) L, Th, AFG, L-AFG, Th-AFG, and L-Th-AFG was evaluated. The checkerboard technique was used to evaluate the synergistic effects of the selected complex, imipenem and vancomycin were used against gram-negative and gram-positive drug-resistant bacteria of ESKAPE pathogens, respectively. MTT colourimetric assays of L, Th, L-AFG, Th-AFG, and L-Th-AFG were performed against fibroblast L929, Hella, and CT26 cell lines. The apoptosis evaluation was carried out according to the manufacturer's instructions using a FITC Annexin V Apoptosis Detection Kit with PI from BioLegend® (San Diego, California).
Results: The results of FTIR and XRD confirmed the incorporation of Th and L into the AFG. Thermogravimetric analysis indicated that the inclusion complex of AFG-Th enhanced the thermal stability of Th significantly. The inclusion complex of Th into the AFG increased its antibacterial activity. Minimum inhibitory concentration of Th was decreased after encapsulation into the AFG. FIC values for Pseudomonas aeruginosa showed additive effects and FIC of other bacteria showed synergism effect. The lowest cytotoxicity was for encapsulated L-AFG against Hella cell line with IC50 of 2755 µg/ml. There was a significance difference between the IC50 of encapsulated Th-AFG against fibroblast L929 and Hella cell line.
Conclusion: The results suggest that AFG-Th can potentially be used as a delivery system for hydrophobic and sensitive compounds to increase their usage in the pharmaceutical and food industries.