Extended Abstract
Introduction
Dermatophytosis is a significant cutaneous disease affecting both humans and animals, with increasing resistance to conventional treatments. Dermatophytes invade keratinized tissues, such as skin, hair, and nails, causing various fungal infections known as tinea, depending on the site of infection. The primary symptoms of the disease are not due to direct fungal invasion of the tissue but rather to the production of secondary metabolites, secretory enzymes, and toxins by the pathogenic fungi, which degrade keratinized tissues and cause damage. Primary drug classes, such as allylamines, polyenes, and azoles, are used topically and systemically for the treatment of dermatophytosis. In recent years, griseofulvin has been used to treat clinical forms of dermatophytosis in Iran; however, limitations, such as limited efficacy, adverse effects, cost-effectiveness considerations, and increasing drug resistance, in common anti-dermatophytic treatments, exist. Therefore, it is logical to plan for the discovery of natural sources containing anti-dermatophyte compounds to potentially overcome these challenges.
Artemisia species (Artemisia sp.), belonging to the Asteraceae family, are widely distributed across various regions worldwide, including Europe, Asia, North Africa, North and South America, and Australia. These plants produce a spectrum of bioactive compounds, such as phenols, terpenoids, sterols, and polyacetylenes, which exhibit antimicrobial, antiviral, anti-inflammatory, and anti-fungal properties. Extracts from different parts of these plants have been traditionally used in Korea and China to treat fever, tumors, malaria, and hepatitis. The present study aimed to determine the anti-fungal activity of petroleum ether, dichloromethane, ethyl acetate, ethanol, and hydroalcoholic extracts of the aerial parts of Artemisia biennis, Artemisia ciniformis, and Artemisia turanica against dermatophyte fungi, including Trichophyton rubrum, Trichophyton verrucosum, Epidermophyton floccosum, and Microsporum cannis.
Methods
This experimental descriptive study examined the petroleum ether, dichloromethane, ethyl acetate, ethanolic, and hydroalcoholic aerial part extracts of Artemisia biennis, Artemisia ciniformis, and Artemisia turanica against fungi causing dermatophytosis.
Fungal Isolates and Culture Media: Three fungal isolates, Microsporum cannis (5069), Trichophyton verrucosum (5056), and Trichophyton rubrum (5143), were obtained from the Iranian Industrial Mycology and Bacteriology Collection Center, affiliated with the Iranian Research Organization for Science and Industry. Epidermophyton floccosum was isolated from patients referring to the Mycology Laboratory of Mahdieh Clinic, Kermanshah University of Medical Sciences. These fungi were cultured on Sabouraud dextrose agar containing chloramphenicol and cyclohexamide (Merck, Germany) for 14 days at 25±2°C. After this period, a suspension of 10⁶ (mL/colony) conidia was prepared from the culture of each fungus.
Plant Species Collection: Various species of Artemisia biennis, Artemisia ciniformis, and Artemisia turanica were collected at the end of the growing season from Zoshak region in the city of Mashhad, Tandoureh National Park in Daregaz County, and Sami’ Abad in Torbat-e Jam County, respectively.
Anti-fungal Effect Evaluation by Culture Media Incorporation Method: For primary screening, plant extracts were dissolved in dimethyl sulfoxide (DMSO). Subsequently, they were mixed with molten Sabouraud dextrose agar containing chloramphenicol and cycloheximide to achieve a final concentration of 1.25 mg/mL. After that, 100 μL of conidia suspension of each fungus (10⁶ mL/colony) was inoculated onto the culture media supplemented with either extract or control, and the tubes were incubated at 25±2°C for 14 days. The anti-fungal effect of each extract was evaluated by observing the complete inhibition of fungal growth.
Evaluation of Minimum Inhibitory Concentration (MIC): The MIC of the extracts was determined using the agar dilution method. Final concentrations of each extract in the culture medium ranged from 39.06~1250 mL/μg. The MIC was determined after a 14-day period at 25±2°C by observing the absence of fungal growth. The DMSO solution (31.25 mL/μL) and terbinafine (2.00~0.0039 mL/μg) were used as negative and positive controls, respectively, in the culture medium. The above experiment was repeated three times for all treatments and controls. Additionally, for each fungus, three pure culture media in three test tubes were prepared as controls to verify fungal growth on Sabouraud dextrose agar containing chloramphenicol and cyclohexamide.
Preliminary Phytochemical Screening: Petroleum ether and dichloromethane extracts were subjected to preliminary phytochemical screening to determine the presence of terpenoids, sterols, and flavonoids using standard procedures.
Sterol Detection: The Liebermann-Burchard reaction was employed to detect sterols. Two mL of acetic anhydride and two drops of concentrated sulfuric acid were added to 3 mL of the chloroform extract. The formation of a blue or green color indicated the presence of steroids.
Terpenoid Detection: An amount of 0.2 g of each extract was dissolved in 6 mL of chloroform and filtered. Concentrated sulfuric acid was then added to the filtrate to form a layer. The development of a reddish-brown color at the interface of the two phases was considered positive for the presence of terpenoids.
Flavonoid Detection: An amount of 0.2 g of each extract was dissolved in 2 mL of ethanol, heated, and filtered. A magnesium metal chip was added to the solution, followed by a few drops of concentrated hydrochloric acid. The appearance of a red or orange color indicated the presence of flavonoids.
Results
Fifteen extracts obtained from the aerial parts of Artemisia biennis, Artemisia ciniformis, and Artemisia turanica exhibited a variable range of inhibitory activity against four dermatophytes: Epidermophyton floccosum, Trichophyton rubrum, Microsporum cannis, and Trichophyton verrucosum.
Epidermophyton floccosum and Microsporum cannis demonstrated the highest resistance (11 out of 15) and sensitivity (12 out of 15), respectively, to the tested extracts. Petroleum ether extracts from all three Artemisia species were the most active extracts employed in the assays, exhibiting inhibitory effects against all four fungi (except for the observed resistance of Trichophyton verrucosum to the petroleum ether extract of Artemisia turanica). Hydroalcoholic extracts exhibited the least anti-dermatophytic activity in the assays. According to the preliminary screening results, various extracts of Artemisia biennis, Artemisia ciniformis, and Artemisia turanica exhibited 15, 14, and 9 cases of inhibition of fungal growth, respectively. Ethyl acetate extracts from all three species demonstrated identical results in priliminary screening so that resistance to these extracts was observed exclusively in Epidermophyton floccosum.
The MIC results demonstrated that all tested fungi were sensitive to all or some concentrations of the extracts used in this experiment, within a concentration range of 39.06 to 1250 mL/μg, as indicated by the absence of fungal growth. Among the extracts used, the lowest MIC belonged to the petroleum ether extract of Artemisia ciniformis at 78.12 mL/μg against Trichophyton rubrum. Additionally, the lowest MIC of Artemisia turanica extracts was found in the petroleum ether extract at 156.25 mL/μg against Trichophyton rubrum and Microsporum cannis. The lowest MIC of Artemisia biennis extracts was determined to be 156.25 mL/μg for the petroleum ether and dichloromethane extracts against Trichophyton rubrum, Microsporum cannis, and Trichophyton verrucosum. On the other hand, the highest inhibitory activity belonged to the petroleum ether and dichloromethane extracts obtained from Artemisia ciniformis and Artemisia turanica. Preliminary phytochemical screening results of the petroleum ether and dichloromethane extracts primarily indicated the presence of sterols and terpenoids.
Conclusion
Some lipophilic compounds present in petroleum ether extracts of Artemisia biennis and Artemisia ciniformis, as well as dichloromethane extract of Artemisia biennis, exhibited significant in vitro concentration-dependent anti-dermatophytic activities.
Ethical Statement
This study was approved by the Research Ethics Committee of Kermanshah University of Medical Sciences (IR.KUMS.REC.1398.080).
Funding
This article has been extracted from the doctoral dissertation of Mr. Abdolmajid Valadbeigi for a specialty degree in Pharmacy at Kermanshah University of Medical Sciences.
Conflicts of Interest
No conflict of interest.
Acknowledgement
The authors would like to thank the financial support provided by the Research and Technology Vice-Chancellor of Kermanshah University of Medical Sciences (No. 980105).
Key message: Some lipophilic compounds present in petroleum ether extracts of Artemisia biennis and Artemisia ciniformis, as well as dichloromethane extract of Artemisia biennis, exhibited significant in vitro concentration-dependent anti-dermatophytic activities.