Advanced Hybrid-Green Ultrasound–Infrared–Microwave Trifolium repens Essential oil Isolation with Multi-Target Ethnomedicine Bioactivity against Neuropathy, Inflammation and Multidrug-Resistant Infection

Sara Al Ayoubi, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon.
Ibrahim Abdelwahab, Department of Microbiology and Immunology, Faculty of Pharmacy, Pharos University in Alexandria, Alexandria, Egypt.
Maha Aboul-Ela, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon
Abdalla El- Lakany, Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
Karim Raafat, Department of Pharmacognosy and Natural Products, Faculty of Pharmacy, Pharos University in Alexandria, Canal El Mahmoudia Street, Beside Green Plaza Complex 21648 Alexandria, EgyptFollow

Keywords

Antimicrobial resistance; Essential oils; Microwave-assisted extraction; Neuropathy; Trifolium repens.

Abstract

Trifolium repens (white clover) is rich in bioactive volatiles with therapeutic potential. This study developed an advanced hybrid ultrasound–infrared–microwave hydro-distillation (UIMHD) method for efficient essential oil (EO) isolation and investigated its active sesquiterpene, pseudoionone, for multi-mechanistic neuroprotection in diabetic neuropathy (DN). The optimized UIMHD system synergistically combined ultrasound cavitation, infrared heating, and microwave hydro-distillation, enhancing yield and reducing time versus conventional extraction. Gas chromatography–mass spectrometry (GC–MS) profiling identified pseudoionone as the dominant constituent. Pharmacological evaluations were performed in alloxan-induced diabetic mice to assess antidiabetic, anti-inflammatory, neuroprotective, and anticonvulsant activities. Cytokine modulation (TNF-α, IL-6, IL-10), oxidative stress markers (CAT, GSH, TBARS), and strychnine-induced seizure assays were conducted to elucidate mechanisms. The UIMHD technique increased T. repens essential oil (TR-EO) yield by 62.5% and shortened isolation time by 50%. Pseudoionone (49.16%) exhibited potent hypoglycemic and insulin-restorative effects, alleviated thermal hyperalgesia and tactile allodynia, and improved antioxidant defenses. Both the TR-EO and pseudoionone significantly down-regulated TNF-α and IL-6 while up-regulating IL-10, indicating anti-inflammatory cytokine balance restoration. Additionally, pseudoionone delayed strychnine-induced tonic seizures, suggesting functional involvement of glycinergic pathways. Strong antibacterial and antibiofilm activities were also observed against multidrug-resistant Shigella dysenteriae. The advanced UIMHD method provides a sustainable platform for isolating bioactive volatiles from T. repens. Pseudoionone emerges as a multi-target natural agent mitigating diabetic neuropathy via TNF-α/IL-6/IL-10 modulation, oxidative-stress restoration, and strychnine-sensitive GlyR potentiation.

Recommended Citation

Ayoubi, Sara Al; Abdelwahab, Ibrahim; Aboul-Ela, Maha; El- Lakany, Abdalla; and Raafat, Karim (2026) "Advanced Hybrid-Green Ultrasound–Infrared–Microwave Trifolium repens Essential oil Isolation with Multi-Target Ethnomedicine Bioactivity against Neuropathy, Inflammation and Multidrug-Resistant Infection," Journal of Food and Drug Analysis: Vol. 34 : Iss. 2 , Article 7.
Available at: https://doi.org/10.38212/2224-6614.3595