Bacterial infections remain a pressing concern for public health globally, with antibiotic resistance compounding the challenge. Despite the advent of antibiotics, bacterial pathogens have evolved various mechanisms to evade the effects of these drugs, leading to the rise and spread of resistant strains. Understanding the dynamics of bacterial infections and antibiotic resistance is crucial for developing effective strategies to combat these threats 1. Recent studies have highlighted the escalating burden of antibiotic resistance and its implications for global health. The World Health Organization (WHO) reports that antibiotic resistance is a growing concern, with multidrug-resistant bacteria causing infections that are increasingly difficult to treat 2.

The future of antimicrobial medication use remains uncertain due to the growing issue of microorganism resistance. Thus, measures to mitigate this issue are required, such as regulating the use of antibiotics, advancing research to comprehend resistance mechanisms at the genetic level, and conducting ongoing investigations to create novel pharmaceuticals, both synthetic and natural. The ultimate goal is to deliver suitable and effective antimicrobial medications to patients 3.

Lactuca virosa, a member of the Asteraceae family, is a biennial herbaceous plant indigenous to Europe and Asia 4, 5. With a rich historical presence in traditional medicine, it has been employed for diverse purposes such as pain relief, sedation, and the treatment of respiratory conditions 6, 7, 8. Several bioactive compounds including lactucin, lactucopicrin and lactucinamide have been identified in L. virosa, contributing to its pharmacological effects. Among these compounds, lactucin and lactucopicrin have been reported to possess antimicrobial properties, prompting interest in exploring the antibacterial activity of L. virosa 9.

The molecular docking tool serves as a pivotal instrument in designing drug structures and predicting binding interactions between ligands and proteins in their three-dimensional structures, thereby elucidating specific activity. Leveraging ligand-based drug design with phytochemicals helps mitigate complexity and streamline the process. By assessing the microbial effects through binding energies of proteins with phytochemicals sourced from L. virosa, novel generation drugs targeting the infection and proliferation of diverse pathogens can be developed 10. The current work aims to screen the phytoconstituents of the L. virosa leaves ethanolic extract using GC-MS analysis and investigate its antibacterial activity.

The phytochemical constituents found in plants play a vital role in their defence mechanisms against various microorganisms, insects, and herbivores 30. In the selected plant investigated, a variety of phytochemicals have been identified, including alkaloids, coumarins, flavonoids, glycosides, phenols, terpenoids, oils and resins. These compounds are summarized in Table 1. The presence of these bioactive compounds suggests their potential involvement in the observed antimicrobial properties of the plants. Alkaloids and phenols are known for their antimicrobial properties due to their ability to disrupt microbial cell membranes or inhibit essential enzymes 31, 32. Flavonoids possess antioxidant and antimicrobial activities 33. The presence of these phytochemical constituents highlights the potential of the selected plant as sources of antimicrobial agents and provides insights into their mechanisms of action against microorganisms.

The ethanolic extract of L. virosa contains thirteen phytochemical constituents as indicated by GC-MS chromatogram analysis (Figure 1). Upon comparison with the NIST library, these constituents were characterized and identified. The bioactive phytocompounds identified in the ethanolic leaf extract exhibit diverse biological activities, as detailed in Table 2. Among the identified compounds, the most abundant compounds are 1-Hexyl-2-Nitrocyclohexane, 1-Hexyl-1-Nitrocyclohexane, 1-Heptacosanol, 2R-Acetoxymethyl-1,3,3-trimethyyl-4t-(3-Methyl-2-buten-1-yl)-1t-cyclohexanol, 1-Hydroxypyrene, and 2,4,4-Trimethyl-3-hydroxymethyl-5a-(3-Methyl-but-2-enyl)-cyclohexene. These compounds are noted for their antibacterial activity, indicating the potential of L. virosa as a source of natural antibacterial agents.

The ethanolic extract of L. virosa contains thirteen phytochemical constituents as indicated by GC-MS chromatogram analysis (Figure 1 ). Upon comparison with the NIST library, these constituents were characterized and identified. The bioactive phytocompounds identified in the ethanolic leaf extract exhibit diverse biological activities, as detailed in Table 2. Among the identified compounds, the most abundant compounds are 1-Hexyl-2-Nitrocyclohexane, 1-Hexyl-1-Nitrocyclohexane, 1-Heptacosanol, 2R-Acetoxymethyl-1,3,3-trimethyyl-4t-(3-Methyl-2-buten-1-yl)-1t-cyclohexanol, 1-Hydroxypyrene, and 2,4,4-Trimethyl-3-hydroxymethyl-5a-(3-Methyl-but-2-enyl)-cyclohexene. These compounds are noted for their antibacterial activity, indicating the potential of L. virosa as a source of natural antibacterial agents.

According to the tabulated docking results presented in Table 3, all five compounds identified through GCMS have effectively docked within the active binding site of the protein domain. Notably, the calculated binding energies for these interactions fall within the range of -6.0 to -9.0 kcal/mol. Among the five evaluated compounds, the docking results highlighted that compounds 1-Hydroxypyrene and 4,4,6A,6B,8A,11,11,14B-Octamethyl-1,4,4A,5,6,6A,6B,7,8,8A,9,10,11,12,12A,14,14A,14B-Octadecahydro-2 exhibited superior binding energies compared to the others. Specifically, these two compounds demonstrated the most favourable binding energies of approximately -8.1 and -8.5 kcal/mol with 6AHT and -8.8 and -8.8 kcal/mol with 5C5H, respectively, based on the molecular docking analyses.

It's worth noting that all compounds comply with the Lipinski rule, as demonstrated in Table 4. However, the specific molecules 2R-Acetoxymethyl-1,3,3-trimethyl-4t-(3-Methyl-2-buten-1-yl)-1t-cyclohexanol and 2,4,4-Trimethyl-3hydroxymethyl-5a-(3-Methyl-but-2-enyl)-cyclohexene stood out as potential candidates for further investigation in drug discovery and development (Table 5). This suggests they have desirable properties that make them worth exploring as potential therapeutic agents.

The Minimum Inhibitory Concentration (MIC) of the L. Virosa leaf extract was assessed against Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus mutans, using concentrations ranging from 0.1 to 0.4 mg/ml. The ethanolic extract of Lactuca virosa showed increasing antibacterial activity with higher concentrations against Bacillus cereus. At 0.1 mg/mL, 0.2 mg/mL and 0.3 mg/mL, the zones of inhibition were 0.8 mm, 0.9 mm and 2.2 mm, respectively. Significant antibacterial activity was observed at highest concentration with 3.3 mm at 0.4 mg/mL. Similarly, the extract showed increasing antibacterial activity with higher concentrations against Streptococcus mutans, with zones of inhibition from 1.0 mm at 0.1 mg/mL to 3.4 mm at 0.4 mg/mL. Antibacterial activity was observed at the highest concentration, with a zone of inhibition of 3.3 mm at 0.4 mg/mL. Tetracycline showed higher zones of inhibition at all concentrations, ranging from 2.8 mm to 3.5 mm. The findings revealed that the extract displayed antibacterial properties against all examined strains, showing efficacy comparable to tetracycline at a concentration of 0.4mg/ml (Table 6). Furthermore, the MIC values revealed that the 0.4mg/ml concentration of the crude ethanol extract effectively inhibited the growth of S. mutans and B. cereus 34. This suggests that the extract may have potential as an antimicrobial agent, particularly against dental caries caused by S. mutans. The research findings suggest that the ethanolic extract of L. virosa exhibits stronger antibacterial activity against Gram-positive bacteria compared to Gram-negative bacteria.

The fact that it showed different antibacterial activity profiles against various strains, with Streptococcus mutans and Bacillus cereus being particularly vulnerable, suggests its potential as a source for novel antimicrobial agents. The presence of alkaloids, terpenoids, flavonoids, and phenols in the preliminary phytochemical screening indicates that these compounds might contribute to the observed antibacterial activity. However, you rightly note that further studies are necessary to fully understand the effectiveness of the crude extracts and to isolate and characterize the specific bioactive compounds responsible for the antibacterial properties. It provides a valuable foundation for further investigation into the potential therapeutic applications of Lactuca virosa and the discovery of new natural bioactive compounds with antibacterial properties.