Identification of chemical compounds, antioxidant potential, and antifungal activity of (Thymus daenensis) essential oil against spoilage fungi causing apple rot

Apple fruit is highly susceptible to fungal spoilage by Penicillium, Botrytis, and Alternaria species. Currently, the use of synthetic fungicides is considered to be the most accessible method of managing and controlling post-harvest diseases of vegetables and fruits, especially apples. However, increasing concern about environmental pollution, the toxicity, and the resistance of fungal pathogens to synthetic fungicides have resulted in an increased demand for less dangerous methods. In this regard, the use of compounds of natural origin that have potential antifungal activity (such as herbal essential oils), can be an effective solution to control and prevent post-harvest diseases of vegetables and fruits. In this study, the potential antifungal activity of Thymus daenensis essential oil was evaluated against fungi species causing apple rot (i.e., Penicillium expansum, Alternaria alternata, and Botrytis cinerea). The chemical compounds, total phenol and flavonoids content, and antioxidant activity of the essential oil were also determined.   In this study, the essential oil of T. daenensis was extracted by the hydrodistillation method and its main chemical compounds were identified and quantified by gas chromatography coupled to mass spectrometry apparatus. Total phenols and flavonoids content of the essential oil were measured using the Folin-Ciocalteu and Aluminum chloride colorimetric methods, respectively. The in-vitro antioxidant activity of T. daenensis essential oil was evaluated based on the DPPH/ABTS free radical scavenging activity, beta-carotene bleaching, and ferric reducing antioxidant power (FRAP) assays. The antifungal effect of the essential oil against Penicillium expansum, Alternaria alternata, and Botrytis cinerea was investigated by the disk diffusion agar (DDA), well diffusion agar (WDA), minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC).   The T. daenensis essential oil was rich in thymol (69.88%), γ-terpinen (8.49%), p-cymene (8.20%), and carvacrol (3.55%). In addition, the total phenol and flavonoids content of the essential oil were 91.45 mg GAE/g and 42.28 mg QE/g, respectively, which had an important role in its antioxidant activity. The T. daenensis essential oil had remarkable DPPH free radical scavenging activity (IC50= 29.30 mg/ml), ABTS free radical scavenging activity (IC50 = 22.68 mg/ml), beta-carotene bleaching inhibitory effect (62.22%), and ferric reducing antioxidant power (30.10 μM QE/g), revealing the electron/hydrogen donating ability of the essential oil. Antifungal results showed that P. expansum was the most sensitive fungi species to the essential oil and lower concentrations of the essential oil were required to inhibit the growth of or kill the species, due to the presence of phenolic compounds (such as thymol and carvacrol) in the oil. Indeed, reactive aromatic nucleus and phenolic OH groups in the structure of phenolic compounds can form hydrogen bonds with –SH groups at the active sites of target enzymes, leading to the deactivation of the fungal enzymes. In addition, the lipophilic nature of the essential oils makes them to be highly absorbed by the lipophilic mycelia and consequently suppress the growth of fungi species. Based on the results, the T. daenensis essential oil could be used as a natural antifungal agent and synthetic fungicide substitute to prevent the growth of pathogenic fungi on apple fruit or other food products and increase their shelf-life.