Phytochemical Characterization and Synergistic Antibacterial Effects of Colebrookea Oppositifolia Essential Oil as Adjuvants to Modern Antibiotics in Combating Drug Resistance

Zifang Shang,1,* Vipasha Sharma,2,* Tarun Kumar,3 Kamal Dev,4,5 Sandip Patil6 1Guangdong Engineering Technological Research Center of Clinical Molecular Diagnosis and Antibody Drugs, Meizhou Academy of Medical Sciences, Meizhou People’s Hospital (Huangtang Hospital), Meizhou, 514031, People’s Republic of China; 2Department of Biotechnology, University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India; 3Mkelly Biotech Pvt Ltd., Mohali, Punjab, India; 4Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India; 5Department of Pharmacology and Toxicology, Wright State University, Dayton, OH, USA; 6Department of Haematology and Oncology, Shenzhen Children’s Hospital, Shenzhen, People’s Republic of China*These authors contributed equally to this workCorrespondence: Sandip Patil; Vipasha Sharma, Email sandippatil1309@yahoo.com; sharma.vipasha@gmail.comBackground: The global threat of multi-drug-resistant bacteria has severely limited the options available for effective antibiotics. This study focuses on the antimicrobial activity and phytochemical characterization of C. oppositifolia extracts, aiming to identify novel plant-based therapeutic agents.Methods: C. oppositifolia specimens-leaves and inflorescence. Specimens were cleaned, sterilized, dried, and ground into a fine powder. Extracts were obtained using methanol and petroleum ether via a Soxhlet apparatus, followed by fractionation with chloroform, n-butanol, and ethyl acetate. Volatile oil was extracted through hydro distillation using a Clevenger apparatus. Phytochemical analysis was conducted to identify bioactive compounds. Biophysical techniques, including UV-visible spectrophotometry, TLC, HPLC, GC-MS, FTIR, and NMR, were employed for characterization. Antimicrobial activity was tested against S. aureus ATCC25922 and E. coli ATCC25922 using agar well and disc diffusion methods, and synergistic effects were assessed with erythromycin and amoxicillin.Results: Methanol extract exhibited bacteriostatic activity with inhibition zones of 13.0 ± 0.2 mm for both S. aureus and E. coli. Petroleum ether, chloroform, n-butanol, and ethyl acetate fractions showed varying inhibition zones. Erythromycin demonstrated bactericidal activity, which was enhanced synergistically when combined with methanol extract and volatile oil, increasing inhibition zones against S. aureus. Phytochemical analysis identified phenols, flavonoids, tannins, coumarins, alkaloids, terpenoids, saponins, and glycosides. FTIR analysis revealed functional groups such as amines, aldehydes, nitriles, alkenes, and sulfones. GC-MS identified 24 compounds, with α-pinene, caryophyllene, and carene as major components. NMR spectra indicated no complex formation between oils and antibiotics, suggesting the compounds act as synergists.Conclusion: The C. oppositifolia extracts possess significant antimicrobial activity and synergistic potential, particularly against S. aureus. The presence of various bioactive compounds suggests a promising role in developing new plant-based therapeutics.Keywords: C. oppositifolia, antimicrobial activity, phytochemical characterization, multidrug-resistant pathogens, synergistic effects