Thymoquinone inhibits germination of dermatophyte arthrospores.

T is an active principle of Nigella sativa (N. sativa), which is a member of the Ranunculaceae family of plants. Several active ingredients have been isolated from the seeds of N. sativa, and many pharmacological effects, such as anti-bacterial, antiparasitic, anti-cancer, and anti-inflammatory have been attributed to them.1 The dermatophytes are a group of fungi, which parasitize the stratum corneum of the skin, hair, and nail causing infections commonly called tinea or ringworm. In infected tissues, the dermatophytes grow in the form of hyphae breaking down by septation into arthrospores. The arthrospores are important morphological forms of dermatophytes for the spread of infection, particularly, via contact with scales exfoliated from infected sites.2 The arthrospores are thus appropriate morphological forms of dermatophytes for the study of anti dermatophyte drugs. In vitro formation of arthrospores can be induced at conditions of increased carbon dioxide tension in the incubation atmosphere, temperature of 37oC, elimination of glucose from the growth medium, or presence of sub-lethal doses of antifungal drugs.3,4 The objective of the present study was to investigate the inhibitory effect of thymoquinone on the germination of dermatophyte arthrospores. The study was conducted in the departments of Dermatology, Pharmacology, and Microbiology, College of Medicine, King Faisal University, Dammam, Kingdom of Saudi Arabia (KSA) from 2005-2006. Approval for the study was obtained from the ethical committee of the King Fahd Hospital of the University (KFHU), Al-Khobar, KSA. Fourteen clinical isolates were obtained from patients attending the dermatology clinic of the KFHU, belonging to various genera of dermatophytes: 4 Trichophyton rubrum, 2 T. violaceum, one T. mentagrophytes var interdigitale, one T. mentagrophytes var mentagrophytes, 3 Microsporum canis, one M. gypseum, and 2 Epidermophyton floccosum, and tried for production of arthrospores. They were sub-cultured as surface lawns on petri dishes under the following conditions: Normal air at 37oC in Dermasel agar (Oxoid); 15% CO2 and 85% normal air at 28 oC in Dermasel agar; 15% CO2 and 85% normal air at 37oC in Dermasel agar; 1% Peptone agar without glucose at 28oC and normal air; and 1% Peptone agar without glucose at 37oC and normal air. One isolate, Trichophyton mentagrophytes (var interdigitale), was selected for the present study because of the abundant formation of arthrospores at 15% CO2 and 85% normal air (Abdullah Hashim Industrial gases and equipments Co. Ltd., Dammam, Saudi Arabia) and a temperature of 37oC after an incubation period of 10 days. Incubation was carried out in a modular incubator chamber (Flow laboratories, Irvine, UK) and gassing the chamber with CO2 and air mixture on a daily basis. Stock cultures were maintained on glucose Peptone agar. The arthrospores suspension was prepared by harvesting surface growth from the petri dish with a scalpel blade, suspending in phosphate buffered saline (PBS) and shaking on a vortex mixer for 5 minutes to separate chains of arthrospores. The suspension was filtered through column type chromatography grade glass wool packed in a 10ml syringe barrel to a depth of 1cm to remove unbroken chains of arthrospores. The filtered arthrospores suspension was washed 3 times in PBS at 3000g for 3 minutes and adjusted to a concentration of 5x106/ml. The viability of the arthrospores was checked before conducting anti-germination assay; 0.3 ml of arthrospores suspension was incubated in 0.9ml of 4% glucose plus 1% peptone broth (SDB) at 37oC for 12 hours on a rotary shaker (100rpm). An arthrospore was considered to have germinated when a visible germ tube had developed. Arthrospores germinated well in SDB, and the viability of arthrospores was 97.8%. Stock solutions of thymoquinone, clotrimazole and griseofulvin, were prepared by dissolving 64 mg of a drug in 50 ml dimethyl sulphoxide (DMSO), allowed to stand for 30 minutes to permit self-sterilization and stored at 4oC. From the stock solutions, the following concentrations of drugs were prepared: Thymoquinone 0.256 mg/ml, 0.128 mg/ml, and 0.064 mg/ml; clotrimazole 0.128 mg/ml; and griseofulvin 0.128 mg/ml. For the germination assay, to 0.3 ml of arthrospores suspension was added 0.3 ml SDB, and 0.6 ml of each concentration of the drugs. Thus, the final concentrations of the drugs in the germination assays were; 0.128, 0.064, and 0.032 mg/ml for thymoquinone, and 0.064 mg/ml for clotrimazole and griseofulvin. Similarly, for the preparation of DMSO control, to 0.3 ml of arthrospores suspension was added 0.3 ml SDB and 0.6 ml of DMSO 10%. Thus, the final concentration of DMSO in the germination assay was 5%, equal to its concentration in the solutions used to prepare 0.128 mg/ml of the drugs. The second control mixture containing 0.6ml of sterile distilled water, 0.3ml arthrospores suspension, and 0.3ml SDB was also prepared. Sets of 3 germination bottles were prepared for each concentration of the drugs and the controls. The Brief Communication