{'fraction_non_alphanumeric': 0.03231479446032095, 'fraction_numerical': 0.019015168168828315, 'mean_word_length': 4.893134715025907, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The Potential Inhibitory Effect of Cuminum Cyminum, Ziziphora Clinopodioides and Nigella Sativa Essential Oils on the Growth of Aspergillus Fumigatus and Aspergillus

The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28oC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2% yeast extract granulated plus 15% Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future.


INTRODUCTION
Moulds of the genus Aspergillus are among the most common fungi in the environment, being found in the air, in the soil, on plants and on decomposing organic matter (22).
Because of their powerful hydrolytic enzymes, fungi can cause a high degree of deterioration when present in/on food-and feed-stuffs (23). Thus, the presence and growth of these fungi in food-and feed-stuffs threatens human and animals health. In immunocompromised subjects, inhalation of spores gives rise to aspergillosis, an invasive infection of the lungs or sinuses and its dissemination to other organs (11).
Resistance of Aspergilli to some clinically used antifungals brings a worrying clinical prognostic in people attacked by aspergillosis (6,9). The wide use and sometimes misuse of antimicrobial agents in both human and animal medicine has been responsible for rapid development of resistant strains, toxicity and drug-drug interactions (16,27).
Regarding the increasing clinical importance given to fungi causing infections and the development of drug resistance, many researchers focused on the antifungal properties of plant products.
Plants from Iranian biomes, such as C. cyminum (Apiaceae; known as Ziree), Z. clinopodioides (Labiatae; known as Avishan) and N. sativa (Ranuculaceae; known as black seed) have been used as natural medicines by local populations in the treatment of several diseases (3, 30).
Previous studies revealed interesting antimicrobial effects from their essential oils (1,15,20). To our knowledge, there is lack of information about their effects on the kinetics of the mycelial growth and germination of Aspergillus conidia. The aims of this study were to evaluate the effect of C. cyminum, Z. clinopodioides and N. sativa essential oils on the growth of two important Aspergillus species, A. fumigatus and A. flavus, recognized as potential air-and/or food-borne pathogens.

Plant materials
The whole aerial parts of C. cyminum, Z. clinopodioides

Extraction of essential oils
Essential oils were isolated by water distillation for 3 h from air-dried materials, using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia (8). The oils were stored at −4°C in sealed brown vials until use. The essential oils were assayed at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods. The stock solutions of oils were prepared according to Souza et al. (29). In this study, all general chemical materials were purchased from Merck Company (Darmstadt, Germany). The fungal strains were precultured on Sabouraud glucose agar slants at 28°C for 10 days. Conidia were taken from the slants Inhibitory effect on the growth of A. fumigatus and A. flavus using sterile distilled water containing 0.5% Tween 80. The resulting mixture of conidia and hyphal fragments was vortexed for 15 s and the heavy particles were allowed to settle for 5 min. The resulting suspension was counted in a Neubauer chamber and standardized to concentrations of 1×10 7 conidia/ml. This suspension was further diluted 1:10 with RPMI 1640 broth to final concentrations of 1×10 6 conidia/ml.

Antifungal activity measurements
The MIC 90 and MFC for 3 essential oils were determined by broth macro-and microdilution methods, according to the

Transmission electron microscopy (TEM)
Fungal materials obtained from 5-day-old cultures of 2% yeast extracted granulated plus 15% Saccharose media and treated with 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml were processed for TEM (5). The samples were pre-fixed with 2.5%

Statistical analysis
Data were analyzed by the unpaired Student's t test. A P value of less than 0.05 was considered statistically significant.

Inhibition of growth
Results of the inhibitory activities of essential oils on the growth of A. fumigatus and A. flavus are presented in Table 1.

Transmission electron microscopy
In untreated fungi (controls), the cell wall was uniform and thoroughly surrounded by an intact fibrillar layer. Plasma membrane was unfolded with a uniform shape and all the organelles, such as nuclei and mitochondria appeared normal ( Fig. 1a and 1b).  Fig. 1c and 1d). Subsequent events were loss of normal conidia and hyphae shape, detachment of fibrillar layer of the cell wall (Fig. 1e, 1f and 1g), destruction of hypha memberanous organelles including nuclei and mitochondria, and finally disorganization of cytoplasmic contents accompanied by intensive degradation and lysis of the nucleus and mitochondria (Fig. 1h). The most remarkable changes in fungal compartments were observed in fungi treated with the highest fungistatic concentrations of the oils (1.5-2 mg/ml).
Destruction and breaking down of plasma membrane at different sites (Fig. 1i), disorganization of conidial and hyphal cytoplasm and complete lysis of membranous organelles seemed to be lead to cells dead ( Fig. 1j and 1k).   however that remains to be proved.
The oils tested, besides the increase in vacuolization, showed a strong alteration in the cytoplasmic membrane. The plasma membrane of Aspergillus species was seen to be irregular and dissociated from the cell wall and cut into small fragments; these membrane segments were dispersed into the cytoplasm. These changes were usually found in fungi treated with imidazole components (26).
Finally, the membranous organelles were disrupted following treatment with oils. The membrane-disruptive activity of essential oil components may be closely associated with the interference with enzymatic reactions of the membrane, such as respiratory electron transport, proton transport, and coupled phosphorylation steps (19). Moreover, a marked depletion of cytoplasmic contents of hyphae accompanied by lysis and disruption of membranes of major organelles, such as nuclei and mitochondria indicated that in high fungistatic concentrations the oils passed not only through the cell wall but also through the plasma membrane and then interacted with membranous structures of the cytoplasmic organelles. The present results clearly showed that the oils were able to inhibit fungal growth by changing the cell uniformity via direct interaction with either cell wall or cytoplasmic membranes. From the observations of TEM, no Inhibitory effect on the growth of A. fumigatus and A. flavus significant morphological changes were found in the hyphae and conidia exposed to the low concentration of the oils, but the conidia exposed to relatively higher concentrations of oils collapsed. There were minor differences in morphological changes of different fungi based on the oils tested. It is considered that this phenomenon is related to constituents of oils that possess a polar functional group like hydroxyl group or have a charged group such as anionic group or cationic group (28).
From the results of this study, it might be concluded that