Paper Details

PJB-2025-213

Network pharmacology and experimental evaluation of singhar Citrus limon peel essential oil for bioactive and antibiofilm properties

 

Asma Ayaz, Adnan Amin, Sajid Ali, Wajid Zaman, Minjun Liang, Yaodong Gu and Yun Seok Heo
Abstract

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Citrus limon peel is a rich source of bioactive monoterpenes that exhibit potential antimicrobial and antioxidant activities. This study aimed to comprehensively characterize the phytochemical composition and biofunctional mechanisms of the indigenous Singhar seedless variety C. limon peel essential oil (EO) by using integrated experimental and in silico approaches. The EO was extracted by hydro-distillation and analyzed via GC–MS. Network pharmacology and molecular docking were employed to elucidate molecular targets and pathways underlying the EO’s antibacterial mechanisms. Antioxidant activity was evaluated using DPPH, FRAP, and H₂O₂ assays, while antibacterial and antibiofilm assays were performed against Staphylococcus aureus strains. GC–MS, revealed 21 compounds, including limonene (61.2%), p-menth-8-en-ol acetate (14.1%), α-terpinene (10.2%), and linalyl anthranilate (9.2%). Network pharmacology identified 71 overlapping targets, including STAT3, MAPK14, and MAPK1, which are enriched in MAPK, IL-17, and VEGF signaling pathways. Docking simulations revealed favorable binding energies (−4.6 to −6.5 kcal/mol), suggesting that EO compounds can disrupt microbial and inflammatory signaling cascades. During experimental validation, the EO demonstrated potent antioxidant activity in a concentration-dependent manner (IC₅₀ = 12.4 µg/mL) and exhibited notable antibacterial efficacy with MIC values ranging from 1.3–21.3 µg/mL, particularly against S. aureus ST-2. Biofilm inhibition reached 77.4% at 2% (v/v), and time-kill kinetics confirmed sustained activity up to 18 h. It was concluded that Singhar C. limon peel EO exerts its bioactivity through multitarget mechanisms, including antibacterial, antibiofilm, and oxidative stress modulation, thus substantiating its potential application as a natural therapeutic agent for managing S. aureus pneumonia infections. Given its food-grade origin, this essential oil also shows strong potential as a natural preservative and antioxidant ingredient for improving food safety and shelf-life.

To Cite this article: Ayaz, A., A. Amin, S. Ali, W. Zaman, M. Liang, Y. Gu and Y.S. Heo. 2026. Network pharmacology and experimental evaluation of singhar Citrus limon peel essential oil for bioactive and antibiofilm properties. Pak. J. Bot., 58(5): DOI: http://dx.doi.org/10.30848/PJB2026-5(18)  
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