Anti Bacterial Activity Of Leucaena Leucephala Seeds Extract Against Pathogenic Bacteria

Abstract

Introducation and aim. The development of urgent therapeutic alternatives is necessary to address the global problem of pathogenic bacteria, which can cause a wide range of diseases, from simple to opportunistic, and are resistant to many antibiotics. The most significant types of these bacteria are Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae.  Medicinal plants are one type of alternative The antibacterial, antioxidant, and anticancer qualities of Leucaena leucocephala seeds have been utilized, and studies have validated their significance in lowering our reliance on antibiotics. The aim was to determined the antibacterial activity of aqueous ,hexan and ethanol extract of L. leucephala seed on s. aureus,  E. coli  and  K. pneumonia Study the synergistic of ethanolic extract and antibiotics (CAZ/ CIP/OFX/) aganisit antibiotic_resistant bacteria.

Material and methods: In the Babylon Governorate, 120 bacterial isolates from various clinical specimens (urin, wounds, and brine) were described in this investigation between September 2024 and February 2025.  Each isolate was cultivated on both MacConkey agar and blood agar. The identification of these isolates was carried out using a combination of methods, including microscopic examination, biochemical assays, and the VITEK automated system. The extracts were analyzed using GC-Ms( Gas Chromatography-Mass Spectrometry) , High-Performance Liquid Chromatography (HPLC) to identify and quantify phenolic compounds, tannins, and vitamin E. The antibacterial activity was assessed using the well diffusion method across a range of concentrations.

Results :three major pathogenic bacteria from 120 clinical isolates collected in Babylon Governorate: S. aureus (50 isolates), E. coli (35 isolates), and K. pneumoniae (35 isolates). S. aureus was prevalent in burn (50%) and wound (50%) samples, and also found in urine (33.33%). E. coli was notably high in urine (35.56%), with lower presence in wound (13.3%) and burn (5%) specimens. K. pneumoniae was exclusively isolated from urine samples (38.89%). Gram staining confirmed 50 positive isolates as S. aureus and 70 negative isolates (35 E. coli and 35 K. pneumoniae). Biochemical tests provided distinct profiles for the identified bacteria. All S. aureus isolates were catalase-positive and showed mannitol fermentation. Conversely, E. coli and K. pneumoniae were catalase-negative. The Indole test was positive only for E. coli, while S. aureus and K. pneumoniae were negative. For the Methyl Red (MR) test, E. coli was positive, and S. aureus and K. pneumoniae were negative. The Citrate test was positive for K. pneumoniae, but negative for S. aureus and E. coli. the antibacterial efficacy of L. leucocephala seed extract using different solvents (aqueous, ethanolic, hexane) against the isolated bacteria. The aqueous extract showed no inhibitory effect against S. aureus or the Gram-negative bacteria (E. coli and K. pneumoniae).

 Ethanolic extract exhibited significant inhibitory effects against S. aureus, with mean inhibition zone diameters ranging from 14.5 mm to 18 mm across various concentrations (25% to 100%). However, neither the ethanolic nor the hexane extracts showed any inhibitory effect against E. coli or K. pneumoniae, suggesting their active compounds may not penetrate Gram-negative bacterial cell membranes. The hexane extract showed limited efficacy against S. aureus, with a slight effect only at 25% concentration (16 mm inhibition zone). Ethanol was identified as the optimal solvent for extracting active compounds with potent antimicrobial properties, particularly against S. aureus. 50 isolates of  S. aureus were tested against 11 common antibiotics. The highest resistance was observed against Cefuroxime (CX) at 54%, followed by Tetracycline (TE) at 30%, and Linezolid (LZ) at 26%. Moderate resistance was seen against Ciprofloxacin (CIP) at 24% and Azithromycin (AZM) at 22%. The isolates showed least resistance to Rifampicin (RA) and Levofloxacin (LEV) at 12% each, and Cefotaxime (CPT) at 6%. Conversely, S. aureus isolates demonstrated high sensitivity to RA (88%), Gentamicin (CN) (84%), Ofloxacin (OFX) (80%), and CPT (74%). Combining the ethanolic extract of L. leucocephala seeds with certain antibiotics showed a synergistic effect against S. aureus. The extract significantly enhanced the activity of Ciprofloxacin (CIP), increasing its inhibition zone diameter from 13 mm to 30 mm, and Ofloxacin (OFX), increasing it from 10 mm to 25 mm. A less pronounced, but still suggestive, synergistic trend was observed with Cefazolin (CAZ), where the inhibition zone increased from 0 mm to 10 mm. This indicates that the extract can augment the effectiveness of certain antibiotics against S. aureus. GC-MS analysis of the ethanolic extract of L. leucocephala seeds identified 12 bioactive compounds, including Methyl linolelaidate, Hexadecanoic acid, methyl ester, and Diisooctyl phthalate. HPLC analysis revealed significant concentrations of phenolic compounds (263.9 mg/100gm) and a lower concentration of tannins (1.58%). Vitamin E was also detected at 0.36%. These compounds likely contribute to the observed antimicrobial activity, particularly the robust efficacy against S. aureus.

Conclusions: This study, conducted in Babylon Governorate, revealed the prevalence of S. aureus, E. coli, and K. pneumoniae as key causative agents in clinical infections. S. aureus was notably prevalent in burn and wound samples, while E. coli and K. pneumoniae were primarily isolated from urine. Traditional methods proved effective for bacterial identification. The ethanolic L. leucocephala seed extract showed potent inhibitory activity against S. aureus but no effect on Gram-negative bacteria. Importantly, the ethanolic extract demonstrated a synergistic effect when combined with certain antibiotics (e.g., ciprofloxacin, ofloxacin) against S. aureus, suggesting enhanced therapeutic potential. Phytochemical analysis confirmed the presence of bioactive compounds in the extract, supporting its therapeutic promise as an antimicrobial or an adjunct therapy against S. aureus infections.