I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
ISSN 0976-6936
In vitro Screening of Nigella sativa Seeds for Antifungal Activity
M. P. Suthar*, P. N. Patel, T. G. Shah, R. K. Patel
1
S. K. Patel College of Pharma Edu & Res., Ganpat University, Mehsana - Gozaria Highway, Kherva,
Ta & Dist : Mehsana, Gujarat, India. 382711
*Corresponding Authors e-mail: paresh_2304@yahoo.co.in
Abstract:
Nigella sativa is an herb from a ranunculaceae
family which is used for the variety of therapeutic
purpose like anticancer, antifungal, antibacterial,
antiparasites and anti-inflammatory. The
mechanism of action of Nigella sativa is
inhibition of DNA synthesis by inhibition of
HDAC enzyme interacting with the chromosome.
The present investigation involves screening of
Nigella sativa seeds plant extracts against the
various pathogenic fungus strains for antifungal
evaluation. Extraction and isolation of Nigella
sativa seeds were carried out using cold
percolation method and these extracts were
evaluated for antifungal activity by standard
broth dilution (NCCLS) method. After evaluating
plant extracts, isolation of Thymoquinone from
plant extracts were carried out by thin layer
chromatographic analysis method and
Thymoquinone was evaluated for antifungal
activity. Thymoquinone was active against most
fungal strains and also proved more active than
standard antifungal drug Amphotericin B.
Key words: mallotus phillipinensis, Rottlerin,
Thymoquinone, EUCAST, NCCLS
1. Introduction
Of the over 100,000 species of fungi, only about
100 species are pathogenic for animals. Fungi
are opportunistic organisms, which are
[1]
ubiquitous in nature . The last two decades
have seen a steady increase in the incidence of
systemic fungal infections especially due to
opportunistic fungi. Prolonged antimicrobial
therapy, invasive procedures,
immunosuppressive therapy and the Acquired
Immunodeficiency Syndrome (AIDS) pandemic
have contributed to the rise in systemic fungal
[2]
infections . There are then many
"opportunistic" fungi which cause infections
almost exclusively in debilitated patients whose
normal defense mechanisms are impaired. The
organisms involved are cosmopolitan fungi
which have a very low inherent virulence.
Currently, there has been a dramatic increase in
fungal infections of this type, in particular
candidiasis, cryptococcosis, aspergillosis, and
zygomycosis. More recently described mycoses
of this category include hyalohyphomycosis and
phaeohyphomycosis. Altogether, some 200
"human pathogens" have been recognized from
among an estimated 1.5 million species of fungi
[3]
. Many of the drugs currently available have
undesirable effects and most of them are
fungistatic and not fungicidal or leads to the
[4, 5]
development if fungal resistance . Hence
there is a real need for a next generation of
safer and more potent antifungal agents. To be
a very effective antifungal drug, it must be
fungicidal rather than fungistatic and have a
broad spectrum of activity, a minimum
emergence of resistant strains, and a selective
mechanism of action. In addition it should have
minimum toxic side effects and good bio-
availability.
The National Committee for Clinical Laboratory
Standards (NCCLS) has set benchmark
methodology by providing laboratory tested
reproducible, consensus peer-reviewed
standards. Different extracts were obtained and
antifungal susceptibilities determined using
NCCLS M27- A2 protocol and the EUCAST
method [6]. The NCCLS M27- A2 protocol is the
benchmark methodology set by The National
Committee for Clinical Laboratory Standards
(NCCLS) and the EUCAST (European
Committee for Antimicrobial Susceptibility
Testing) method is the standard method used by
European countries. It is similar to micro dilution
assay to the NCCLS method, but uses a larger
inoculum size and higher glucose concentration
in the medium as well as a spectrophotometric
[7]
endpoint determination . NCCLS is now
referred as CLSI (Clinical and Laboratory
84
 I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
Standards Institute). The end-point for present
investigation was determined by visual
determination using turbidity levels as stated in
the NCCLS M27-A2 protocol, the
spectrophotometric determination using a plate
reader, and the colorimetric determination using
[8]
an oxidationreduction indicator- resazurin .
Chemical constituent of Nigella sativa volatile oil
Contains in the % Range (W/W) of
Thymoquinone 27.8, Carvacrol 5.8-11.6 , P-
cymene 15.5-31.7, -pinene 9.3, 4-terpineol 2-
6.6, Longifolene 1-8, t-anethole 0.25-3,
Reduction product of thymoquinone. 16. [9- 14].
The seeds of N. sativa have been subjected to a
range of pharmacological investigations in
recent years. These studies have showed a
wide spectrum of activities such as antibacterial
[15, 16] [17] [18, 19]
, antitumor , anti-inflammatory , CNS
depressant and analgesic , hypoglycemic [21],
[20]
smooth muscles relaxant [22- 24] cytotoxic and
[25]
immunostimulant . Some of these activities
have been predominantly attributed to the
volatile and fixed oils. To the best of our
knowledge antifungal activity of the fixed and
volatile oils obtained from the seeds of N. sativa
has not been the subject of much study. The
antifungal activity of various extracts of Nigella
sativa seeds were investigated on different
species such as Aspergillus niger and various
Candida species by the standard agar plate
method but not by the standard broth
microdilution method. Therefore, We decided for
anti-fungal screening of various extracts of the
Nigella sativa on the various fungal strains by
the standard broth dilution method (NCCLS) as
well as some modification in NCCLS method
which used sphectrophotometric determination
of the end point.
2. Materials
Ethanol, 3-(N-morpholino) propanesulfonic acid
(MOPS), Resazurin, Czapek yeast extract agar,
Amphotericin B, Dimethyl sulphoxide (DMSO).
The fungus strains used for the study were
obtained from the Microbial Type Culture
Collection (MTCC, India). The fungus strains
used in the study are Aspergillus fumigatus
(2550), Aspergillus flavus (871), Candida
albicans (183), Candida tropicalis (184),
Candida parapsilosis (1744), Issatchenkia
orientalis (3020), Cryptococcus albidus var.
albidus (2661), Cryptococcus layrentii var.
laurentii (2898).
ISSN 0976-6936
Collection of plant material: The plant material
(Nigella sativa seeds) was collected from the Ms
Lallubhai Vrajlal Gandhi and sons, (Ahmedabad,
Gujarat, India) and authenticated by the Bio-
science department of Ganpat University.
3. Methods
Nigella sativa seeds plant extracts were
prepared by cold percolation method. Three
extracts were prepared and evaluated for
cytotoxic and antifungal activity. The three
extracts prepared were: A. Methanolic extract,
B. Ethanolic extract and C. Water extract.
Methanolic extract: Methanolic extract of the
Nigella sativa seeds was prepared by socking
150 gm finely grounded powder of Nigella sativa
seeds in 150 ml of methanol for 7 days. After 7
days, the extract was filtered through What man
filter paper and evaporated till dryness.
Ethanolic extract: Ethanolic extract of the
Nigella sativa seeds was prepared by simple
socking 50 gm of finely grounded powder of
Nigella sativa seeds in sufficient amount of
ethanol for 24 hours. After 24 hours, the extract
was filtered through what Mann filter paper and
the residual matter was again soaked with
sufficient amount of ethanol for 24 hours. After
24 hours, the extract was filtered through
Whatman filter paper and then was combined of
two extract and evaporated till dryness.
Water extract: Water extract were prepared by
using Clevenger apparatus. 25 gm of Nigella
sativa seeds were boiled with 50 ml of distilled
water. The volatile constituent of Nigella sativa
evaporated and collected in small vials and
stored in refrigerator until use.
Thin layer chromatography of Nigella sativa:
Each of the above extracts and the solution of
reference standard of Thymoquinone were
spotted individually on precoated silica gel G60
F 254 TLC plates. Plates were developed in
Diethyl ether : Benzene (1:1) as solvent system
to resolve different components of these
extracts. The identification of the thymoquinone
spot, obtained from the extracts of Nigella sativa
is essentially identical to thymoquinone
standard.
86
 I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
Antifungal susceptibility test using standard
method of NCCLS (The National Committee for
Clinical Laboratory Standards)
A. Preparation of broth medium: 10.4 gm of
RPMI-1640 medium supplemented with
glutamine and phenol red, without bicarbonate
and 34.53 gm 3-(N-morpholino) propanesulfonic
acid (MOPS) was dissolved in 400 ml of distilled
water. pH was adjusted to 7.0 at 25 C with 1
mol/L sodium hydroxide. The volume was made
up to 0.5 L with water and was filtered, sterilized
and was stored at 4C until required.
B. Preparation of inocula: Fungal strains were
sub-cultured on to their respective growth
medium and incubated for 48 hrs at 25-30C.
From these plates, several colonies were
transferred to 5 ml of sterile distilled water. The
suspensions were mixed for 15 s to ensure
homogeneity and were subsequently diluted to
match the turbidity of a 0.5 McFarland standard
(i.e. OD = 0.120.15 at = 530 nm,
6
corresponding to 15 X 10 CFU/ml). Then the
working suspensions were prepared by a 1 in 30
further dilution of the stock suspension in sterile
3
distilled water to yield 15 X 10 CFU/ml. 0.1ml
sterilized solution of resazurin (20 mg/ml in
water) was supplemented to the working
suspension.
C. Preparation of samples: Stock solutions of
the plant extracts and the positive control drug
amphotericin B were prepared in dimethyl
sulphoxide (DMSO) at the concentrations of 100
mg/ml. Further it was diluted to 1:50 in broth
D. Preparation of plates: Microdilution
susceptibility testing was performed in flat-
bottom 96-well clear plates containing broth
medium (50l) in each well. Sample solutions
(50l) were subsequently serially diluted two-fold
in the plates with the broth, starting with the final
concentration of 5000 mg/L. The working
inoculum suspension (50l) was added to give a
final inoculum concentration of 0.52.5 X
10 3CFU/ml. Amphotericin B was used as the
standard antifungal drug. Sterility and growth
controls in the presence of DMSO were also
included. The plates were then incubated at
37C for 48 h. The amount of growth was
measured using plate reader at =450nm.
Antifungal susceptibility test using standard
method of EUCAST (European Committee for
Antimicrobial Susceptibility Testing)
ISSN 0976-6936
A. Preparation of broth medium: 10.4 gms of
RPMI-1640 medium supplemented with
glutamine and phenol red, without bicarbonate
and 34.53g 3- (N-morpholino) propanesulfonic
acid (MOPS) was dissolved in 400 ml of distilled
water. pH was adjusted to 7.0 at 25 C with 1
mol/L sodium hydroxide. The medium was
further supplemented with glucose (18 g) to
achieve a final concentration of 2% glucose
(w/v). The volume was made upto 0.5 L with
water and was filtered, sterilized and was stored
at 4C until required.
B. Preparation of inocula: Fungal strains were
sub-cultured on to their respective growth
medium and incubated for 48 hrs at 25-30C.
From these plates, several colonies were
transferred to 5 ml of sterile distilled water. The
suspensions were mixed for 15 s to ensure
homogeneity and were subsequently diluted to
match the turbidity of a 0.5 McFarland standard
(i.e. OD = 0.120.15 at = 530 nm,
6
corresponding to 15 X 10 CFU/ml). Then the
working suspensions were prepared by a 1 in 10
further dilution of the stock suspension in sterile
distilled water to yield 15 X 105 CFU/ml.
C. Preparation of samples: Stock solutions of
the plant extracts and the positive control drug
amphotericin B were prepared in Dimethyl
Sulphoxide (DMSO) at the concentrations of 100
mg/ml. Further it was diluted to 1:50 in broth
D. Preparation of plates: Microdilution
susceptibility testing was performed in flat-
bottom 96-well clear plates containing broth
medium (50ml) in each well. Sample solutions
(50ml) were subsequently serially diluted two-
fold in the plates with the broth, starting with the
final concentration of 5000 mg/L. The working
inoculum suspension (50ml) was added to give
a final inoculum concentration of 0.52.5 X
10 5CFU/ml. Amphotericin B was used as the
standard antifungal drug. Sterility and growth
controls in the presence of DMSO were also
included. The plates were then incubated at
37C for 24 h. The amount of growth was
measured using plate reader at =450nm.
Plate assignment: Plates were prepared as
shown in figure and then subjected to result
determination after appropriate time. For
EUCAST method the results were obtained with
use of the plate reader whereas for NCCLS
method the visual readings as well as
87
 I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
ISSN 0976-6936
spectrophotometric readings were also extract. (fraction-3). 4. Water extract.
considered. (fraction-4)

4. Results And Discussion
After the development of the TLC plate, it was
found that the extracts were separated in 4
different zones where thymoquinone appeared
at Rf 0.82. Reference standard of thymoquinone
also showed only one spot at the Rf of 0.82.
(A)
Each extracts was tested against fungal strains
in dose dependent manner (Concentration from
100 g-0.005 g). End point was determined
sphectophotometrically by ELISA plate reader.
NCCLS test replicates for anti-fungal activity
were analyzed every 24 hr for three days to
determine the percentage growth inhibition in
the presence of the extracts. The percentage
growth inhibition of extracts against each fungus
strains was calculated for the 24 hr, 48 hr, and
72 hr. Methanolic, ethanolic and water extract
(50l) were serially diluted two-fold in
concentration range of 100 to 0.005 g/ml in
plates with the Cryptococcus laurentii. The
working inoculums suspension (50l) was added
to give final inoculums concentration of 0.52.5
X 10 3CFU/ml. The plates were incubated at 37
C for 48 h. The amount of growth was
measured using plate reader at =450nm.
Percentage inhibition of extract against all cell
line was calculated using the following formula.

% Cell survival = (AT-AB) / (AC-AB) X 100

Where,
AT=Absorbance of treated cells (drug) at
different concentration.
AB=Absorbance of blank (without cell)
AC=Absorbance of control (untreated)
There by,
% Cell growth inhibition = (100-% cell
survival)
(B)
The effects of extracts were expressed by IC50
values calculated from dose response curves.
IC50 C. C. C. C. C. C.tropica A. A.
(g/ml) layrent albidu albica ori parap lis fumiga flavus
ii s ns ent silosis tus
alis
Metha 17.13 26.03 61.46 12. 42.71 > 100 46.44 54.65
nolic 61
extract
Ethano 26.03 > 100 > 100 > 41.02 86.95 67.88 53.24
lic 10
extract 0
Water 29.08 22.85 50.71 14. 50.64 > 100 66.35 > 100
extract 57
Ampho 57.68 21.86 27.65 22. 15.50 69.29 69.84 89.26
tericin 26
B
Thymo 9.313 20.83 23.33 25. 2.308 >100 23.40 >100
quinon 33
e
(C)
Fig 1: A) Chromatographic finger printing Table 1: IC50 values of plant extracts and
of the plant extracts. B) Chromatographic Amphotericin B
analysis at UV 254 nm. C) Chromato
graphic analysis at UV 366 nm. Chromato Methanolic extract showed most potent activity
graphic finger printing of plant extracts. against Issatchenkia orientalis with IC50 value
1. Thymoquinone standard. (fraction-1), 2. 12.61 g/ml, while Amphotericin B has IC50
Methanolic extract. (fraction-2), 3. Ethanolic value 22.26 g/ml. Methanolic extract also
88
 I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
ISSN 0976-6936
shown significant activity against Cryptococcus
laurentii, Cryptococcus albidus with IC50 value
17.13 g/ml and 26.03 g/ml, respectively.
Ethanolic extract shown significant activity
against Cryptococcus laurentii with IC50 value
26.03 g/ml and proved more potent than
Amphotericin B with IC50 value 57.68 g/ml.

Ethanolic extract does not shown activity against

Cryptococcus albidus, Candida albicans and
Issatchenkia orientalis where Amphotericin B
showed activity against these fungus strains with
IC 50 value 21.86 g/ml, 27.65 g/ml, and 22.26
g/ml, respectively.

Water extract showed significant activity against

Issatchenkia orientalis with IC50 value 14.57
g/ml and also proved to be more potent than
Amphotericin B. Water extract does not showed
activity against fungal strain Candida tropicalis
while Amphotericin showed antifungal activity
against Candida tropicalis with IC50 value 69.29
g/ml.

Plant extracts showed antifungal activity against

most pathogenic fungi except Candida tropicalis
and Aspergillus flavus and Thymoquinone also
showed antifungal activity against most
pathogenic fugal strains. Thymoquinone showed
antifungal activity against most fungal strains. It
also showed comparative antifungal activity with
standard drug Amphotericin B. Thymoquinone
showed most potent activity against Candida
parapsilosis with IC50 value 2.308 g/ml and
Cryptococcus laurentii with IC50 value 9.313
g/ml while Amphotericin B showed activity
against these two strains with IC50 value 57.68
g/ml and 15.50 g/ml, respectively and proved
more potent than Amphotericin B.
Thymoquinone also exhibited activity against
Cryptococcus albidus, Candida albicans,
Isstchenkia orientalis, and Aspergillus fumigates
with IC50 value 20.83 g/ml, 23.33 g/ml, 25.33
g/ml and 23.40 g/ml. Interestingly,
Thymoquinone showed antifungal activity
against most fungal strains except Candida
tropicalis and Aspergillus flavus. Amphotericin B
showed more antifungal activity with IC50 value
22.26 g/ml while Thymoquinone showed
antifungal activity with IC50 value 25.33 g/ml
against Isstchenkia orientali.

89
 I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
department of S. K. Patel College of Pharma
Edu & Res., Ganpat University for providing
facility for my research work.
References
Fig 2: Activity of Methanolic , Ethanolic and
Water extracts of Nigella sativa seeds
against various pathogenic fungal species
Fig 3: Activity of Thymoquinone against
various pathogenic fungal species
5. Conclusion
From the results obtained, it can be concluded
that methanolic extract shown most potent
antifungal activity against Cryptococcus laurentii
and Cryptococcus albidus, Issatchenkia
orientalis and Aspergillus fumigatus and doesnt
shown significant activity against Candida
tropicalis. Ethanolic extract shown activity
against Cryptococcus laurentii, Issatchenkia
orientalis and Aspergillus species. Water extract
shown significant activity against Cryptococcus
laurentii, Cryptococcus albidus, Candida
albicans, Issatchenkia orientalis and Aspergillus
fumigatus. Thymoquinone was found to be
present in all three extracts. Thymoquinone was
active against most fungal strains except
Candida tropicalis and Aspergillus flavus and
also proved more active than standard
antifungal drug Amphotericin B.
Acknowledgement :-
Authors are thankful to pharmaceutical
biotechnology department and pharmacognosy
ISSN 0976-6936
1. Betty M. Partridge, M. A. Athar, H. I.
Winner , Chick embryo inoculation as a
pathogenicity test for Candida species, J
Clin Pathol 1971;24:645-648
2. W. Chierakul, A. Rajanuwong, V.
Wuthiekanun, N. Teerawattanasook, M.
Gasiprong, A. Simpson, W. Chaowagul
and N. J. White, The changing pattern of
bloodstream infections associated with
the rise in HIV prevalence in
northeastern Thailand , Transactions of
the Royal Society of Tropical Medicine
and Hygiene, 2004, 98, 11, 678-686
3. Emilie Fralle, Christophe Nol, Nicole
Nolard, Franoise Symoens, Maria-Sueli
Felipe, Eduardo Dei-Cas, Daniel
Camus, Eric Viscogliosi and Laurence
Delhaes, Molecular Phylogenetics and
Evolution, Manganese superoxide
dismutase based phylogeny of
pathogenic fungi, 2006; 41, 1, 28-39
4. K.Hazen, Fungicidal versus fungistatic
activity of terbinafine and itraconazole:
An in vitro comparison Journal of the
American Academy of Dermatology, 38,
5, S37-S41
5. Nicolas Venisse, Nicolas Grgoire,
Manuella Marliat, and William Couet,
Mechanism-Based Pharmacokinetic-
Pharmacodynamic Models of In Vitro
Fungistatic and Fungicidal Effects
against Candida albicans, Antimicrobial
Agents and Chemotherapy, 2008; 52,
3, 937-943
6. Manjuan Liu , Veronique Seidel, David
R. Katerere and Alexander I. Gray,
Colorimetric broth microdilution method
for the antifungal screening of plant
extracts against yeasts , Methods ,
2007; 42, 4, 325-329
7. A. Espinel-Ingroff, F. Barchiesi, M.
Cuenca-Estrella,M. A. Pfaller,M. Rinaldi,
J. L. Rodriguez-Tudela and P. E.
Verweij , International and Multicenter
Comparison of EUCAST and CLSI M27-
A2 Broth Microdilution Methods for
Testing Susceptibilities of Candida spp.
to Fluconazole, Itraconazole,
Posaconazole, and Voriconazole , J Clin
Microbiol. 2005; 43, 8, 38843889
90
I J PA S International Journal of Pharmaceutical and Applied Sciences/1 (2)/2010
8. Vishnu Chaturvedi, Rama Ramani and
Michael A. Pfaller , Collaborative Study
of the NCCLS and Flow Cytometry
Methods for Antifungal Susceptibility
Testing of Candida albicans, Journal of
Clinical Microbiology, 2004; 42, 5,
2249-2251
9. M. A. Mansour, O. T. Ginawi, T. El-
Hadiyah, A. S. El-Khatib, O. A. Al-
Shabanah, H. A, effects of volatile oil
constituents of Nigella sativa on carbon
tetrachloride -induced hepatotoxicity in
mice: evidence for antioxidant effects of
thymoquinone, Res. Commun. Mol.
Pathol. Pharmacol, 2001; 110: (3&4);
239-251,
10. Bahman Nickavara,, Faraz Mojaba,
Katayoun Javidniab, andMohammad Ali
Roodgar Amolia , Chemical
Composition of the Fixed and Volatile
Oils of Nigella sativa L. from Iran , Z.
Naturforsch. 2003; 58c, 629-631
11. Nagi, Mahmoud N. and Hussain A.
Almakki , Thymoquinine (main
constituent of volatile oil of Nigella
sativa) supplementation induces
quinone reductase and glutathione
transferase in mice liver: possible role in
protection against chemical
carcinogenesis and toxicity,
Phytotherapy Research , YEAR 23, 9,
12951298
12. H. Hosseinzadeh, S. Parvardeh, M. Asl,
H. Sadeghnia, T. Ziaee , Effect of
thymoquinone and Nigella sativa seeds
oil on lipid peroxidation level during
global cerebral ischemia-reperfusion
injury in rat hippocampus,
Phytomedicine, YEAR 14, 9, 621-627
13. M. Akram Khan , Chemical composition
and medicinal properties of Nigella
sativa Linn. , Inflammopharmacology,
1999; 7, 15-35
14. Ferdous A.-J., Islam S.-N., Ashan M.,
Hasan C.-M., and, Ahmed Z.-U., In vitro
antibacterial activity of the volatile oil of
Nigella sativa seeds against multiple
drug resistant isolates of Shigella, V.
Cholerae and E. coli. Phytother. Res.,
1992; 6, 137-140
ISSN 0976-6936
15. Hanafy M.-S. and Hatem M.-E., Studies
on the antimicrobial activity of Nigella
sativa seed (black cumin). J.
Ethnopharmacol., 1991; 34, 275-278.
16. Rathee P.-S., Mishra S.-H., and Kaushal
R., Anti microbial activity of essential
oil, fixed oil and unsa- ponifiable matter
of Nigella sativa L. Indian J. Pharm. Sci.,
1982; 44, 8-10
17. David R.-W., Omar A.-G., and Peter A.-
C., The in vitro antitumor activity of
some crude and purified components of
black seed. Nigella sativa. Anticancer
Res.,1998;18, 1527-1532
18. Houghton P.-J., Zarka R., Heras B., and
Hoult J.-R.-S., Fixed oil of Nigella sativa
and derived thy- moquinone inhibit
eicosanoid generation in leuko- cytes
and membrane lipid peroxidation. Planta
Med., 1995; 61, 33-36.
19. Mutabagani A. and El-Mahdy S.-A.,
Study of the anti-inflammatory activity of
Nigella sativa L., and thymoquinine in
rats. Saudi Pharm. J., 1997; 5, 110-
113.
20. Khanna T., Zaidi F.-A., and Dandiya P.-
C., CNS and analgesic studies on
Nigella sativa. Fitoterapia, 1993; 64,
407-410
21. Al-Hader A., Aqel M., and Hasan Z.,
Hypoglyce mic effects of the volatile oil
of Nigella sativa. Int. J. Pharmacogn.,
1993; 31, 96-100
22. Aqel M. , Effects of Nigella sativa seeds
on intesti- nal smooth muscle. Int. J.
Pharmacogn., 1993; 31, 55-60.
23. Aqel M., The relaxing effects of the
volatile oil of Nigella sativa seeds on
vascular smooth muscles. Dirasat.,
1995; 19, 91-100.
24. Aqel M. and Shaheen R., Effects of the
volatile oil of Nigella sativa seeds on the
uterine smooth muscle of rat and guinea
pig. J. Ethnopharmacol., 1996; 52, 23-
26.
25. Swamy S.-M.-K. and Tan B.-K.-H.,
Cytotoxic and immunopotentiating
effects of ethanolic extract of Nigella
sativa L. seeds. J. Ethnopharmacol.,
2000; 70, 1-7.
91