1. AN ALTERNATIVE TREATMENT WITH NIGELLA SATIVA EXTRACTS WITH A SYNERGICAL EFFECT WITH CLASSICAL THERAPY
 Authors: Andrei Bita1, Alexandra Floriana Roşu2, Daniela Călina3, Lucica Roşu4, Ovidiu Zlatian4, Cristina Dindere2, Adrian Simionescu5
1 Student, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, Romania
2 Student, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania
3 Clinical Pharmacy department, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania
4 Microbiology department, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania
5 University of Craiova, Romania

2. Background
Data from literature shows an increasing resistance of fungi and parasites involved in human infections to commonly used antifungal/antiparasitary agents. For these reasons, efforts are made to obtain new therapeutics agents with increased efficacy, obtained from medicinal plants.
Nigella sativa (black cumin, Kalonji) is an annual herbaceous plant growing in the Mediterranean region for its seeds which contain 40% fixed oil, and up to 1,4% volatile oil.
The seeds extracts showed pharmacological effects such as antidiabetic, spasmolytic and bronchodilator, antioxidant hepatoprotective, antihyperlipemic, analgesic and anti-inflammatory effects in various studies.
The extracts also showed in vitro and in vivo antimicrobial, antileishmanial and anticestodal effects.
At least in part these effects may be due to an active component of the volatile oil from the seeds, thymoquinone.
We decided to study the effects of aqueous, methanolic, and chloroformic extracts of the seeds on fungal strains, compared with those of a classical antifungal currently used in therapy of those infections (Clotrimazole).

3. Material and methods
By the means of classical mycological diagnosis methods, we isolated and identified 20 Candida albicans strains from pathological products (pharingeal exudates and vaginal secretions) sampled from patients from different departments (Pediatrics, Intensive Care, Obstetrics -Gynecology).
The extractions: Aqueous and methanol extractions were performed by the following method: 50g of black seed powder were used with 300 ml of water or methanol for hours. The extracts were filtered using filter paper and the solvents were evaporated using rotary distillation apparatus. In order to obtain a completely dry extract, the resultant extracts were transferred to glass dishes and were left at 50°C for 24 hours. Then, they were left at 4°C until assessment of their antifungal activities.
Chloroformic extraction: 600 g of ground black seed in 1500 ml chloroform were incubated at 25°C for one week, during which shaking was carried out up to 5 times a day. The resultant solution was filtered and dried.

4. Material and methods
Nigella sativa seeds: The seeds were collected from medicinal herbal drugs shop.
Chemicals: Methanol and chloroform were purchased from Merck Company, and Clotrimazole disk from standard antifungal susceptibility testing kits (Becton – Dickinson).
The methanolic and chloroformic extracts were diluted (34,12,8,4,2 µg per disk) and the aqueous extracts were not diluted. Paper disks made of paper filter Whattman no. 1 with 6 mm diameter were tainted with the extracts, then dried and sterilized with ultraviolet light.

5. Material and methods
Simultaneously, we performed the antifungal activity assessment against clotrimazole disks (Becton-Dickinson)
The disks were stored at 4° C and used in antifungal susceptibility test by disk diffusion Kirby – Bauer method.
The Candida albicans strains were isolated on Sabouraud medium. It was effectuated the identification by culture, morphological, biochemical, pathogenic characteristics.
The antifungal effect was evaluated by measuring the diameter of the inhibition zones, accounting for the concentration of extracts in each disk.

6. Results
The results of the tests were interpretated by measuring the diameters of the fungal growing’s inhibition zones. The antifungal activity of the extracts were expressed through two symbols “-” (without antifungal effect) and “+” ( the inhibition of the growing).
The fungal cultures on Sabouraud medium with a diameter of inhibition areas around the discs higher that 7 mm were considered susceptible to the antifungal action of tested substances.
The fungal cultures with a diameter around the discs smaller than 7 mm were considered resistant.

7. Results Candida albicans colonies on Sabouraud culture media
Methanolic extract µg/disk/Zone of inhibition(  mm)
Chlorphormic extract µg/disk/Zone of inhibition ( mm)
Aqueous extract µg/disk/Zone of inhibition (  mm)
Clotrimazole (µg)/Zone of inhibition ( mm)
34/12 (disk 3)
34/10 (disk 8)
34/0 (disk 2)
8/18 (disk 1)
12/12 (disk 4)
12/9,5 (disk 9)
8/10 (disk 5)
8/8 (disk 10)
4/10 (disk 6)
4/8 (disk 11)
2/8 (disk 7)
2/7 (disk 12)
Antifungal activity of methanolic, chlorophormic and aqueous extracts of Nigella sativa
Candida albicans colonies on Sabouraud culture media
Candida albicans inhibition by the Nigella sativa extracts

8. University of Medicine and Pharmacy
Conclusions
The methanolic and chlorophormic extracts of Nigella sativa had pronounced antifungal effects on Candida albicans strains, comparable with those of Clotrimazole.
The study offers the perspective of a successfull alternative therapy with natural products, as an addition to classical therapy in fungal infections.
University of Medicine and Pharmacy
Craiova, Romania