Cinnamaldehyde and Eugenol against Candida: an in vitro study

The main finding is that cinnamaldehyde and eugenol were the most active compounds in the studied system. Both inhibited all 18 strains. For cinnamaldehyde, the average zone of inhibition around the disc was 69 mm, the average MIC was 50.05 mg/L, and the minimum fungicidal concentration, or MFC, was 109.26 mg/L. The MFC indicates the amount required to prevent fungal regrowth in the subsequent test. For eugenol, the average values were 35.2 mm, 455.42 mg/L, and 690.09 mg/L.

In the Candida albicans strain selected for the time-kill assay, eugenol eliminated viable cells within one hour and cinnamaldehyde within four hours. Together, 20.48 mg/L of cinnamaldehyde and 41.40 mg/L of eugenol yielded an FICI of 0.625. Since an FICI between 0.5 and 1 indicates additivity, the data show that in this model, doses could be reduced while maintaining inhibition. However, it does not demonstrate synergy in the strict sense, which the authors define as an FICI equal to or less than 0.5. Alpha-pinene showed minor activity; limonene and eucalyptol did not provide comparable results.

This research is relevant because it addresses a specific experimental question: can multiple aromatic components have different antifungal activity compared to isolated substances? Here, the answer is limited to cultures: the cinnamaldehyde-eugenol pair showed an additive effect against the strain used in the combination test. This supports the idea that the chemical composition of a mixture may matter, without turning it into proof of treatment.

The data relates to a possible biological pathway: when the yeast membrane loses integrity or the availability of ergosterol—a molecule essential for the structure of the fungal membrane—changes, the cell may lose internal components and fail to proliferate. Independent studies have explored this pathway in Candida models [1]. However, it remains necessary to distinguish a signal measured in a test tube from an effect in human tissue, where absorption, metabolism, local irritation, and immune system response radically change the context.

This is an in vitro study, meaning it was conducted on microorganisms cultured in a laboratory. It does not include human subjects, does not compare a therapy against a placebo, and does not measure recovery, recurrence, symptoms, or clinical safety. Therefore, it does not allow for the conclusion that the mixture prevents or cures intestinal, vaginal, oral, or invasive candidiasis.

The combination test was performed on the Candida albicans strain 15; it is therefore not a demonstration applicable to all species and strains. Furthermore, the tests were conducted in duplicate, a factor that limits the precision of the estimates. The absence of an effect on Lactobacillus acidophilus at the tested concentrations is a favorable preliminary signal in the model, not proof of selectivity within the human gut microbiota. Finally, the mixture was additive and not synergistic: using these terms as equivalents would make the result appear stronger than what was reported by the authors.

A first key point concerns the fungal membrane. Khan and colleagues studied a strain of Candida albicans in vitro using electron microscopy, flow cytometry, and biochemical tests. Cinnamaldehyde and eugenol damaged the cell wall and membrane and, at half of their respective MIC, reduced ergosterol biosynthesis by 67.41% and 76.23%. This result supports the transition from molecular exposure to loss of cellular integrity hypothesized in Saracino's study; the confirmation is mechanistic and moderate, but based on a single strain and not on a human organism [1].

A second study examined two strains of Candida albicans, including pre-formed biofilms, which are adherent microbial communities that are more difficult to control. Eugenol and cinnamaldehyde reduced the biofilm and showed favorable interactions with fluconazole; FICI against biofilms ranged up to 0.14 for eugenol and 0.312 for cinnamaldehyde in specific comparisons. This does not verify the same mixture as the main study, but it confirms that both molecules can remain active in a more complex experimental condition. The limitation is the absence of clinical data [2].

Alves and colleagues then evaluated cinnamaldehyde against multiple Candida species, multispecies biofilms, and human cells in culture. They observed fungicidal and anti-biofilm activity; computer simulations suggested possible interactions with proteins involved in the membrane. This evidence is indirect regarding the association with eugenol: molecular docking only indicates that a molecule could fit into a protein zone; it does not prove that the binding occurs in the organism. The level of confirmation therefore remains experimental [3].

Results are not uniform across every species, combination, and model. De Sousa and colleagues examined 24 strains of Candida auris in vitro. Eugenol and cinnamaldehyde had a fungicidal effect, but the combination of each with itraconazole proved to be antagonistic or showed no detectable interaction. Furthermore, cinnamaldehyde was cytotoxic to Vero cell lines at concentrations active against the yeast, whereas eugenol was not in the same system. This does not refute the additive effect observed by Saracino and colleagues, but it shows that the response depends on the associated compound, the fungal species, and the cellular model [4].

In a subsequent study on Candidozyma auris biofilms, eugenol and cinnamaldehyde reduced biomass in most clinical isolates, but metabolic activity increased in the majority of isolates during treatment. The authors proposed, without definitively proving it, the existence of heteroresistance, i.e., the presence of cell subgroups with different sensitivities. This finding suggests that one should not equate biomass reduction with complete biofilm inactivation. No directly conflicting evidence sufficiently relevant to the same cinnamaldehyde-eugenol mixture against the same strains has been identified; this does not prove the absence of contrary results [5].