Document Type : Research Paper
Authors
Department of Biological Sciences and Technologies, Faculty of member, Yadegar-e-Imam Khomeini (RAH) Share Rey Branch, Islamic Azad University, Tehran, Iran
Abstract
Background and objectives: Nepeta binaludensis Jamzad is a rare, aromatic, and endemic species known for its antibacterial activity, attributed to its phenolic and flavonoid content. Given the rising issue of antibiotic resistance, exploring innovative therapeutic approaches, such as utilizing plant-derived components in conjunction with nanoparticles, is essential. These methods enhance plants' antibacterial properties while reducing the quantity of these valuable resources needed, thereby contributing to their conservation. The current study investigated the antimicrobial properties of a silver nanoemulsion derived from Nepeta binaludensis extract. It also investigated its effect on the expression of the toxA gene, which is involved in Toxin A secretion, and the algD gene, which plays a role in biofilm formation, in Pseudomonas aeruginosa.
Methodology: The Nepeta binaludensis flowering aerial parts were collected from Binaloud Mts in the northeast of Razavi Khorasan Province at an altitude of 2517 m. The ethanolic extract of the species was obtained from 40 g of the plant-dried powder using the maceration method. The extract's flavonoid and phenolic content were measured using an HPLC apparatus. This study used a silver nanoemulsion-loaded plant extract that was previously prepared by the high-energy method. The synthesis accuracy of the nanoemulsion was evaluated using FTIR, SEM, DLS, Zeta potential, and XRD techniques. The antibacterial effects of varying concentrations of silver nanoparticles, crude extract, and silver nanoemulsion were assessed against the standard bacterial strain Pseudomonas aeruginosa using the Kirby-Bauer disk diffusion test and the microdilution plate technique. RNA extraction from the bacteria was performed utilizing treatments that included the minimum inhibitory concentration (MIC) and sub-minimum inhibitory concentration (subMIC) of silver nanoparticles, the crude extract, and the silver nanoemulsion containing the extract. Following cDNA synthesis, the expression changes of the toxA and algD genes were analyzed using Real-time PCR. The outcomes of the Real-time PCR were assessed with REST software. The results were analyzed using one-way ANOVA with Microsoft Excel 2016 and GraphPad Prism 9.5.1, with a P value of less than 0.05.
Results: The HPLC analysis of the extract composition indicated that the plant extract did not contain gallic acid, catechin, rutin, or quercetin. Among the phenolic constituents, including chlorogenic acid, caffeic acid, salicylic acid, and rosmarinic acid, rosmarinic acid exhibited the highest abundance, measuring 20.82 mg/g. The evaluation of the synthesized nanoemulsion confirmed that the particles are spherical with a diameter of 270 nm. The surface charge is negative, and the particles are non-crystalline, and the presence of water and ester bonds has been detected. The Kirby-Bauer disk diffusion test results indicated that the smallest inhibition zone (12 mm) was associated with silver nanoparticle treatment. Conversely, the largest inhibition zone (21 mm) was observed with the treatment involving silver nanoemulsion of the extract, which was approximately equivalent to the inhibition zone induced by the antibiotic ceftazidime (CAZ, 30µg). The minimum values for MBC, MIC, and Sub MIC were 80, 40, and 20 µg/ml, respectively, corresponding to the silver nanoemulsion containing the extract. The algD gene expression level decreased by a factor of 0.87 when treated with silver nanoemulsion of the extract (40µg/ml), compared to the control group (P < 0.01). The toxA gene expression level was reduced by a factor of 0.78 following treatment with the silver nanoemulsion of the extract (P < 0.05). The changes in gene expression induced by varying concentrations of plant crude extract and silver nanoparticles did not show a significant difference when compared to the control group.
Conclusion: The nanoemulsion of the plant extract exhibits a greater growth inhibitory effect and enhanced bactericidal activity. Given that the algD gene plays a crucial role in biofilm formation and the toxA gene is responsible for the secretion of the endotoxin A, it appears that the silver nanoemulsion may effectively inhibit growth by influencing the expression of genes in Pseudomonas aeruginosa. The reduction in droplet size within the nanoemulsion enhances the concentration of bioactive compounds and antibacterial properties compared to the crude extract. Consequently, this makes it suitable for applications in the pharmaceutical and food industries.
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