Investigating the interactive efficacy of the enantiomers of Limonene

Abstract

Limonene is a widely distributed compound present in many essential oils. This cyclic monoterpene has been well studied, but what has been often overlooked is the possible influence of stereochemistry on biological activity, particularly in combination with other essential oil compounds. This study aimed to investigate the in vitro antimicrobial inhibition (minimum inhibitory concentration–MIC and anti-quorum sensing–QS), and toxicological screening (brine shrimp lethality assay–BLSA) of the enantiomers of Limonene, alone and in 1:1 combinations with 14 essential oil compounds. When investigated independently, the enantiomers were broadly comparable (MIC 0.25–4.00 mg/mL; MQSIC 0.38–0.50 mg/mL; ≤5.8% mortality at 48 h). However, variations in the enantiomeric pair were evident when investigated in combination. S-(–)-Limonene displayed the most synergy (ΣFIC ≤0.50), in combination with β-caryophyllene against Cryptococcus neoformans (MIC = 0.13 mg/mL; ΣFIC = 0.50). R-(+)-Limonene reduced the concentration required to block violacein production, lowering the MQSIC to 0.06 mg/mL when paired with Estragole or Geraniol (ΣFQSIC = 0.19–0.31). R-(+)-Limonene formed non-toxic combinations with γ-Terpinene and α-Terpineol (ΣFPM ≤0.23). The chirality of Limonene affected both efficacy and toxicity in one-third of the combinations investigated, demonstrating that stereoselectivity is critical, yet frequently overlooked, in natural product formulation.