Thymoquinone, an active principle of Nigella sativa, inhibited Aspergillus niger

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Pakistan J. Med. Res.
Vol. 42, No. 3, 2003
Thymoquinone, an active principle of Nigella sativa,
ihibited Aspergillus niger
Al-Jabre S*, Al-Akloby OM*, Al-Qurashi AR**, Akhtar N**, Al-Dossary A*, Randhawa MA***
Department of *Dermatology, ** Microbiology and ***Pharmacology, College of Medicine, King Faisal
University, Dammam, Kingdom of Saudi Arabia
Summary
Nigella sativa (N. Sativa) seed called as ‘Habbah Al-Sauda’ in Arabic, is commonly used as a natural
remedy for many ailments over 2000 years and frequently added to bread and prickles as a
flavouring agent. Recently, many active principles have been isolated from N. Sativa including
thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellidine,
nigellimine-N-oxide and alpha-hedrin. Besides many other pharmacological effects, activity of N.
sativa oil, ether extract and its active principle, thymohydroquinone, has been reported in the
literature against a number of bacteria (including Staphylococcus aureus, Pseudomonas aeruginosa
and Escherichia coli) and yeast (Candida albicans). Therefore, we thought that N. sativa or some of
its active principles might have useful action against Aspergillus as well, which commonly causes
opportunistic infection in immune deficient individuals. We studied the growth of Aspergillus niger
in dermacel agar containing 0.25, 0.5, 1 & 2 mg/ml of thymoquinone; drmasel agar with selective
supplement and dermasel agar alone. There was 0, 25, 77.1 and 100% inhibition of A. niger with
0.25, 0.5, 1 & 2 mg/ml of thymoquinone on day 7, giving MIC as 2 mg/ml. We hope our study will
promote further investigations to determine usefulness of thymoquinone or N. sativa in the treatment
and prevention of Aspergillosis.
Introduction
N. sativa, one of the Ranunculaceae, commonly grows in the Middle East, Eastern Europe and
Middle Asia. In Arab countries it is commonly known as ‘Habba Al-Sauda’ or ‘Habba Al-Baraka’. N.
Sativa seed is frequently added to bread and prickles as a flavouring agent and used as a natural
remedy for many ailments over 2000 years 1, 2.
Recently many active principles have been isolated from N. sativa, including thymoquinone,
thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellimine-N- oxide,
nigellidine and alpha-hedrin 3, 4, 5, 6, 7 & 8. Over the last few decades, a number of pharmaco-therapeutic
effects of various extracts of N. sativa and some of its active principles have been investigated, using
modern scientific techniques, and recently reviewed by Randhawa and Al-Ghamdy 9.
Anti-bacterial effect of the phenolic fraction of N. sativa oil was first reported by Topozada et al10.
Thymohydroquinone was later isolated 4 and found to have high activity against gram-positive
microorganisms. Later, the diethyl-ether extract of N. sativa was reported to inhibit Staphylococcus
aureus, Pseudomonas aeruginosa and Escherichia coli. It also showed synergistic effect with
streptomycin & gentamycin and additive effect with spectinomycin, erythromycin, tobramycin,
doxycycline, chloramphenicol, nalidixic acid, ampicillin, lincomycin and co-trimoxazole. In
addition, the extract also inhibited a pathogenic yeast, Candida albicans11. Recently, crude extracts of
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N. sativa were reported to have a promising effect on multi-resistant organisms including gram-
positive and gram- negative bacteria12. In view of the wide spectrum of anti-microbial activity of N.
sativa we thought it might have useful activity against some fungi, like Aspergillus, besides Candida
albicans.
Aspergilli comprise a group of rapidly growing hyaline moulds that are widely distributed in the
environment and are common contaminants of clinical laboratories. They commonly cause
opportunistic infection in humans. Aspergillosis may present as well defined clinical syndromes
involving a variety of sites and organ systems: pulmonary, disseminated, central nervous system,
cutaneous, endocardial and nasoorbital. It almost exclusively occurs in patients who are immuno-
suppressed or neutropenic, particularly in transplant recipients and those with leukemias or
lymphomas. Aspergillosis is also reported in corticosteroid use, intravenous drug abuse and later
stages of AIDS. Of the some 700 Aspergillus species described, only 19 have been cited as causing
human infections; of these only 4 are recovered with any frequency in hospitalized patients:
Aspergillus fumigatus, A. flavus, A. niger and A. terreus 13.
Materials and Methods
Strains of A. niger were isolated from contamination in petri-dishes being used for some culture
sensitivity tests in the Department of Microbiology, College of Medicine, King Faisal University,
Dammam. The specimens were sub-cultured on Dermasel agar (Oxoid) and incubated at 30o C for 7
days. The growth was identified as A. niger by microscopy after staining with lactophenol cotton
blue.
Thymoquinone was dissolved in small amount of sterile distilled water and then mixed with
sufficient presterilized dermasel agar to obtain 2 mg/ml of thymoquinone. Serial dilutions were then
made to get thymoquinone 1, 0.5 and 0.25 mg/ml.
The isolates of A. niger were subcultured on three sets of culture media. Set 1: dermasel agar only,
Set 2: dermasel agar with dermasel selective supplement (containing cycloheximide 200 mg/500 ml
and chloramphenicol 25 mg/500 ml) and Set 3: dermasel agar with 0.25, 0.5, 1 & 2 mg/ml of
thymoquinone. Four petri-plates were inoculated in each set.
A mycelial disc, 5 mm in diameter, cut from the periphery of 5-7 day old cultures of A. niger in
dermasel agar was aseptically inoculated onto different sets of media. The inoculated plates were
incubated at 30o C. The cultures were examined on the 3rd, 5th and 7th day and results interpreted by
visual impression as well as by measurement of the mean diameter of the growth of Aspergillus14.
The percentage inhibition of A. niger with different concentrations of thymoquinone was then
calculated by taking its growth in non-drug dermasel agar as 100%. The growth of A. niger in
dermasel agar with the selective supplement was taken as active control.
Results
There was 0, 25, 77.1 and 100% inhibition of A. niger with 0.25, 0.5, 1 & 2 mg/ml of thymoquinone
on day 7, giving MIC as 2 mg/ml (table 1), while with the selective supplement the inhibition was
only 32.7 %. A. niger covered the entire area of perti-plates (9 cm diameter, taken as 100%) when
inoculated in dermasel agar alone on day 3 to 7 .
The mean diameter of the growth of A. niger in dermasel agar containing various concentrations of
thymoquinone and dermasel agar with selective supplement is given in table 2a & 2b, respectively.
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Discussion
N. Sativa has been used for many ailments in ancient Greek, Indian and Arabic medicines. For
example, Ibne-Sina recommended the use of N. sativa for the treatment of various microbial
infections of skin (warts, collar-stud abscess and other abscesses, chronic fungal infections such as
ring worm), eye and gastrointestinal tract as well as for the expulsion of worms from the intestines15.
In the present study we observed a dose related anti-fungal effect of thymoquinone, an active
principal of N. sativa; 0.25, 0.5, 0.1 and 0.2 % thymoquinone inhibited 0, 25, 77.1 and 100 % of A.
niger with 0.25, 0.5, 1 & 2 mg/ml of thymoquinone on day 7. Earlier, 25-400 micrograms of diethyl-
ether extract per disc of N. sativa seed has been reported to inhibit a concentration dependent growth
of Gram-positive and Gram-negative bacteria11. Moreover, inhibition in the growth of coliforms,
staphylococci and yeasts/moulds in the processed cheese spread by 0.1 - 0.3% hexane-extracted N.
Sativa oil has also been reported 16.
There was some growth of A. niger in dermasel agar with selective supplement on day 3, 5 & 7 as
shown in table 2b. Which shows that the selective supplement (containing cycloheximide 200
mg/500 ml and chloramphenicol 25 mg/500 ml) failed to completely inhibit the growth of A. niger.
We hope our study will promote further investigations to determine usefulness of N. sativa in the
treatment and prevention of Aspergillosis and possibly other local and systemic fungal infections in
immuno-compromised patients.
Conclusion
Thymoquinone, an active principle of Nigella sativa, effectively inhibited the growth of Aspergillus
niger (MIC 2mg/ml).
Acknowledgments:
We gratefully acknowledge Dr. Mastour S. Al-Ghamdi, Vice Dean (Academic Affairs), College of
Medicine, King Faisal University, Dammam, Saudi Arabia, for the provision of thymoquinone base
for our study.
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Table 1. Percentage inhibition of the growth of Aspergillus niger with different
concentrations of thymoquinone on Day 3, 5 and 7.
Thymoquinone
mg/ml
% inhibition of A. niger growth
Day 3 Day 5 Day 7
0. 25 25 Nil Nil
0. 5 76.8 55.4 25
1 100 91.8 77.1
2 100 100 100
Table 2. Growth of Aspergillus niger in dermasel agar containing different concentrations of
thymoquinone and in dermasel agar with selective supplement.
a. Dermasel agar with thymoquinone
Concentrations
mg/ml
Mean diameter + SD (cm) of A. niger growth (n = 4)
Day 3 Day 5 Day 7
0.25 +++(*) ++++(**) ++++
0.5 2.09 + 0.2 4.01 + 0.49 +++
1 Nil 0.74 + 0.43 2.06 + 0.8
2 Nil Nil Nil
b. Dermasel agar with selective supplement
Mean diameter + SD (cm) of A. niger growth (n = 4)
Day 3 Day 5 Day 7
2.05 + 0.11 4.35 + 0.39 6.06 + 0.11
* Growth of A. niger covering ¾ of the petri-plate
** Growth of A. niger completely covering the petrti-plate (9 cm), taken as 100%.
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