# Synergistic potential and apoptosis induction of Bunium persicum essential oil and its pure components, cuminaldehyde and γ-terpinene, in combination with fluconazole on Candida albicans isolates: in vitro and in silico evaluation

## TL;DR

This study shows that combining Bunium persicum essential oil and its components with fluconazole can enhance antifungal effects and induce cell death in Candida albicans.

## Contribution

The study demonstrates the synergistic and apoptotic potential of natural compounds with fluconazole against resistant Candida isolates.

## Key findings

- Cuminaldehyde showed the lowest minimum inhibitory and fungicidal concentrations compared to other agents.
- Fluconazole combined with cuminaldehyde had the highest synergistic effect (63.6%) against Candida isolates.
- Molecular docking confirmed that cuminaldehyde and γ-terpinene interact with lanosterol 14-alpha-demethylase.

## Abstract

Fluconazole resistance in Candida species is on the rise, posing a significant clinical challenge. There is a growing interest in using complementary therapies, especially those
 from natural sources. This study aimed to evaluate the synergistic and apoptotic effects of Bunium persicum essential oil (BPEO) and its two pure components,
cuminaldehyde (CA) and γ-terpinene (γ-TPN), combined with fluconazole (FLC) on susceptible and resistant C. albicans isolates. Moreover, molecular docking was used to study the interactions between lanosterol 14-alpha-demethylase and each agent.

The BPEO was prepared using the Clevenger apparatus and the hydro-distillation method. The in vitro antifungal activity was evaluated according to the Clinical and
Laboratory Standards Institute guideline (M60). The checkerboard and isobologram assays assessed the interaction between BPEO, CA, γ-TPN, and FLC.
The necrotic and apoptotic effects of different agents were analyzed using a flow cytometry assay. An in-silico study was performed to examine the receptor-ligand interaction.

The CA showed the lowest minimum inhibitory concentrations and minimum fungicidal concentrations, compared to BPEO and γ-TPN. Statistical analyses indicated significant differences between
resistant and sensitive C. albicans isolates regarding minimum inhibitory concentration values of BPEO, CA, and γ-TPN. The most synergistic effect was
obtained for FLC combined with CA (n=7, 63.6%), followed by BPEO (n=6, 54.5%), and γ-TPN (n=3, 27.2%). Statistical analyses indicated the synergistic effect of FLC in
combination with CA was more than γ-TPN (p=0.023). Apoptotic indicators confirmed that the tested compounds could cause cell death in yeast cells.
Combination of each natural component with FLC resulted in a greater apoptosis effect than each tested agent alone. The docking study indicated that both pure compounds have
interactivity with the protein residue of 14α-demethylase.

The results indicated that the synergistic properties of natural products combined with synthetic antifungal agents available in the market could contribute to developing effective therapeutic strategies, particularly in resistant fungal species.