First Investigation of Phytochemical Screening, HPLC-MS Characterization, and Antibacterial Activity of Azadirachta indica A. Juss (Mim or Neem) Leaf Extracts, Grown in the Republic of Chad
Authors: Ousman Brahim Mahamat , Mennane Zakaria , Boussaoudi Ibtissam , Otchom Brahim Boy , Saoud Younes 
Published: February 2025

Objective: This study aims to investigate the phytochemical screening, characterize the chemical composition, and determine the antibacterial activity of Azadirachta indica A. Juss (known in Chad as Mim or Neem) leaf extracts from the Republic of Chad. Methods: Leaf extracts were obtained using Soxhlet extraction. Phytochemical screening was carried out through qualitative tests. The chemical composition was characterized using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Antibacterial activity was tested using the agar diffusion method, and the inhibitory zone diameters were measured. Minimum inhibitory concentrations and minimum bactericidal concentrations were determined using the microdilution method. The bacterial strains tested included Gram+: Staphylococcus saprophyticus, Methicillin-resistant Staphylococcus sp., and Staphylococcus aureus; as well as Gram-: Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Escherichia coli, Escherichia coli extended-spectrum beta-lactamase, and Salmonella sp. Results: The qualitative tests indicated the presence of several compounds, including phenolics, flavonoids, saponins, terpenoids, steroids, carbohydrates and alkaloids, all known to exhibit antibacterial properties. The HPLC-MS analysis of the ethanol leaf extracts identified diethyl phthalate as the major compound, comprising 92.31% of the extract. Additionally, minor compounds were detected, including phytol at 1.68%. Diethyl phthalate is an organic compound well-known for its significant antimicrobial properties. In the HPLC-MS analysis of the water leaf extracts, several promising phenolic compounds were revealed. These included butylated hydroxytoluene (BHT) at 28.12%, benzoic acid at 5.61%, 2-methoxy-4-vinylphenol at 3.45%, eugenol at 2.12%, vanillin at 1.17%, 1,2-benzenediol at 0.62%, and phenylacetic acid at 0.46%. The HPLC-MS analysis of methanol leaf extracts revealed major effective compounds, including benzenamine N N-dimethyl- (14.69%), phytol (7.75%), 1,1,1,5,5,5-hexamethyl-3-((trimethylsilyl)oxy) trisiloxane (7.52%), linolenic acid methyl ester (ME) (6.73%), palmitic acid (ME) (hexadecanoic acid) (6.72%), and bistrimethylsilyl N acetyl eicosasphinga-4,11-dienine (4.91%). The study showed significant antibacterial activity against all tested Gram-positive strains, with inhibition zone diameters (IZDs) ranging from 10 ± 1.00 mm to 15 ± 1.00 mm. The minimum inhibitory concentration (MIC) values varied from 0.06 to 0.50 mg/mL, while the minimum bactericidal concentration (MBC) values were higher than 0.5 mg/mL. No antibacterial activity was detected against any Gram-negative strains. Conclusion: The detected compounds may have significant therapeutic applications. Furthermore, the observed antibacterial activity against Gram-positive strains supports the traditional medicinal uses of this plant in the Republic of Chad.