Antibacterial Activity of Total Extracts and Essential oil of Nigella Sativa L. Seeds in Mice

Abstract

The purpose of this study was to evaluate the antibacterial activity of total crude extracts and essential oil (EO) of Nigella sativa L. seeds in mate mice infected intraperitoneally with Staphylococcus aureus or Escherichia coli (0.1mL from 106 colony forming units/ml suspension). After 24 hours, the infected mice were subjected to different doses of TE or EO or received 33 mg/kg of gentamicin (a positive control) or 0.4 mL of normal saline (a negative control). After 24 hours, aspirated specimens from intraperitoneal fluids were cultured on a soybean casein digest agar plate surface. The inhibitory effect of the methanol extract at a dose of 2.14 g/kg in mice infected with S. aureus was 87.5%. The doses of 1.2 and 2.14 g/kg in mice infected with E. coli were 100% compared with mice who received saline (the negative control). While the aqueous extract did not show any inhibitory effect on either micro-organism, the effect of the chloroform extract at dose of 2.6 g/kg and 33 mg/kg gentamicin (the positive control) was 100 %. The EO at dose of 0.3 g/kg in mice infected with S. aureus and E. coli showed 100% inhibitory effect compared with mice who received saline. N. sativa methanol and chloroform seed extracts as well as its essential oil have dose dependent antibacterial activities on the Gram-positive and Gram-negative organisms.

Full text

Pharmacolgyonline 2: 429-435 (2007) Hosseinzadeh et al
429
Antibacterial Activity of Total Extracts and Essential oil
of Nigella Sativa L. Seeds in Mice
Hossein Hosseinzadeh1, B. S. Fazly Bazzaz2, and Maryam Motevaly Haghi3
1-Correspondence author: Pharmaceutical Research Center, Faculty of Pharmacy, 1365-
91775, Mashhad University of Medical Sciences (MUMS), Mashhad, I.R. Iran. Fax: 98511
8823251,
E-mail: hosseinzadehh@mums.ac.ir or @gmail.com
2- Department of Pharmaceutical Microbiology, Bu Ali Biotechnology Research Center and
School of Pharmacy, MUMS, Mashhad, I.R.Iran.
3- School of Pharmacy and, MUMS, Mashhad, I.R.Iran.
ABSTRACT
The purpose of this study was to evaluate the antibacterial activity of total crude extracts and
essential oil (EO) of Nigella sativa L. seeds in male mice infected intraperitoneally with
Staphylococcus aureus or Escherichia coli (0.1mL from 106 colony forming units/ml
suspension). After 24 hours, the infected mice were subjected to different doses of TE or EO
or received 33 mg/kg of gentamicin (a positive control) or 0.4 mL of normal saline (a
negative control). After 24 hours, aspirated specimens from intraperitoneal fluids were
cultured on a soybean casein digest agar plate surface. The inhibitory effect of the methanol
extract at a dose of 2.14 g/kg in mice infected with S. aureus was 87.5%. The doses of 1.2 and
2.14 g/kg in mice infected with E. coli were 100% compared with mice who received saline
(the negative control). While the aqueous extract did not show any inhibitory effect on either
micro-organism, the effect of the chloroform extract at dose of 2.6 g/kg and 33 mg/kg
gentamicin (the positive control) was 100 %. The EO at dose of 0.3 g/kg in mice infected with
S. aureus and E. coli showed 100% inhibitory effect compared with mice who received saline.
N. sativa methanol and chloroform seed extracts as well as its essential oil have dose
dependent antibacterial activities on the Gram-positive and Gram-negative organisms.
KEYWORDS: Nigella sativa, Black cumin, antibacterial activity, Staphylococcus aureus,
Escherichia coli.
INTRODUCTION
Nigella sativa Linn. is indigenous to the
Mediterranean region but has been cultivated
into other parts of the world including Saudi
Arabia, northern Africa and parts of Asia. The
plant is known by names, such as black cumin
(English), blck-caraway seeds (USA) and
shonaiz (Persian) (Khan, 1999). Different
pharmacological effects such as isulinotropic
(Fararh et al., 2002), hypoglycemic (El-
Dakhakhny et al., 2002), anticancer (Mabrouk
et al., 2002; Salomi et al., 1992),
antinociceptive, anti-inflammatory (Abdel-
Fattah et al., 2000; El-Dakhakhny et al., 2002;
Ghannadi et al., 2005), hepatoprotective
(Mahmoud et al., 2002), neuroprotective
(Kanter et al., 2006a), antihistamine, antiulcer
(Kanter et al 2006 b) and bronchodilator
(Gilani et al., 2001) activities have been
reported for this plant. Black cumin has been

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traditionally used in the Indian continent,
Arabian countries and Europe for culinary
and medicinal purposes as a natural remedy
for a number of illnesses and conditions that
include asthma, hypertension, diabetes,
inflammation, cough, bronchitis, headache,
eczema, fever, dizziness and influenza. The
seeds or its oil are used as a carminative,
diuretic, lactagogue and vermifuge (Ali and
Blunden, 2003).
N. sativa seeds contain 36%–38% fixed oils,
proteins, alkaloids, saponin and 0.4%–2.5%
essential oil. The fixed oil is composed
mainly of unsaturated fatty acids. The
essential oil was analyzed using GC-MS.
Many components were characterized, but the
major ones were thymoquinone (27.8%–
57.0%), ρ-cymene (7.1%–15.5%), carvacrol
(5.8%–11.6%), t-anethole (0.25%–2.3%), 4-
terpineol (2.0%–6.6%) and longifoline
(1.0%–8.0%). Thymoquinone readily
dimerizes to form dithymoquinone. Four
alkaloids have been reported as constituents
of N. sativa seeds. Two, nigellicine and
nigellidine have an indazole nucleus, whereas
nigellimine and its N-oxide are isoquinolines
(Ali and Blunden, 2003).
As the public becomes more interested in
herbal medicine and bacterial pathogens
become more resistant to commercial
antibiotics, scientists are increasingly
investigating the antibacterial properties of
plant extracts and fractions (Fazly Bazzaz et
al., 1997; Hassanzadeh et al., 2001; Taskova
et al., 2002; Kariba, 2002). Antimicrobial
activities have been reported for the extracts
of N. sativa seed in vitro (Hanafy and Hatem,
1991; Morsi, 2000). To extend this activity
and show this effect in a body, the
antibacterial activity of crude extracts and
essential oil of N. sativa seed were studied on
infected mice with Staphylococcus aureus and
Escherichia coli.
METHODOLOGY
Animals
Male BALB/c mice (25 ± 3 g) were obtained
from a random bred colony of maintained
with laboratory pellet chow (Khorassan
Javane Co, Mashhad, I.R. Iran) in animal
house of Mashhad University of Medical
Sciences. Animals were housed in a colony
room with a 12/12 hour light/dark cycle at 24
± 1 °C. All animal experiments were carried
out in accordance with Mashhad University of
Medical Sciences, Ethical Committee acts.
Plant materials
N. sativa seeds were purchased from Gonabad
(Khorasan Province). The methanol extract
(ME) of seeds was prepared using Soxhlet
apparatus. The aqueous extract (AE) was
decocted with hot water for 15 min. The
chloroform extract (CE) was macerated with
chloroform for 72 hour. The extracts were
then concentrated under reduced pressure to
volume desired. The residual water was
evaporated to dryness at 30 °C on a water
bath. In this study ME, CE and AE were
called total extract (TE). Essential oil (EO)

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was prepared by hydrodistilation (Samsam-
Shariat and Moatar, 1996).
Test microorganisms
The strains of bacteria used in this study were
Staphylococcus aureus (ATCC 29737) and
Escherichia coli (ATCC 8739).
All bacteria were cultured from soybean
casein digest agar (Merck) stock cultures
already at 4°C into the fresh prepared agar
and cultured overnight at 35° C. The bacterial
suspensions were prepared (106 cfu/ml) using
normal saline from these cultures.
Assay procedure
240 mice were used in this study. 30 groups
of animals were used, each of which
contained 8 mice. Each group of eight mice
was infected with 0.1 ml (106 cfu/ml) of S.
aureus or E. coli suspension intraperitoneally.
Twenty four hour later, each group received
different treatments. Groups were designed as
following:
Negative control: Group 1 received 0.4 ml
normal saline against S. aureus and group 2
received 0.4 ml normal saline against E. coli.
Positive control: Group 3 received 33 mg/kg
gentamicin against S. aureus and group 4
received 33 mg/kg gentamicin against E. coli.
ME: Groups 5-7 received 0.3, 1.2 and 2.14
g/kg the methanol extract against S. aureus,
respectively and groups 8-10 received 0.3, 1.2
and 2.14 g/kg the methanol extract,
respectively against E. coli CE: Groups 11-13
received 0.38, 1.5 and 2.6 g/kg the chloroform
extract, respectively against S. aureus and
groups 14-16 received 0.38, 1.5 and 2.6 g/kg
the chloroform extract, respectively against E.
coli
AC: Groups 17-19 received 0.24, 0.96 and
1.68 mg/kg the aqueous extract, respectively
against S. aureus and groups 20-22 received
0.24, 0.96 and 1.68 mg/kg the aqueous
extract, respectively against E. coli
EO: Groups 23-26 received 0.03, 0.12, 0.21
and 0.3 g/kg the essential oil, respectively
against S. aureus and groups 27-30 received
0.03, 0.12, 0.21 and 0.3 g/kg the essential oil,
respectively against E. coli
After 24 hours, 0.1 ml aspirated specimens
from intraperitoneal fluid were cultured on
soybean casein digest agar (Merck) plate
surface. Plates were incubated overnight at
35° C and the number of colonies formed on
each plate was counted.
Statistical analysis
The resulting colonies were counted and
expressed in terms of percent colonies formed
from each aspiration sample. This number
was compared with the negative control group
and tested with Fisher’s exact test. The p-
values less than 0.05 were considered to be
statistically significant.
RESULTS
Gentamicin, as positive control, completely
inhibited the appearance of colonies of both

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micro-organisms (Table 1-4). The methanol
extract showed significant antibacterial
activity against both micro-organisms. The
effect of this extract was more significant on
E. coli (Table 1). The chloroform extract also
showed significant antibacterial activity
Table1. Effects of intraperitoneal injection of methanol extract (ME) of N. sativa seed on infected
mice with micro-organisms.
Treatment Dose %Activity
E.coli S. aureus
Normal saline 0.1 ml 0 0
Gentamicin 33 mg/kg 100*** 100***
ME 0.3 g/kg 87.5** 50
ME 1.2 g/kg 100*** 75**
ME 2.14 g/kg 100*** 87.5**
** P< 0.01, *** P< 0.001, compared with normal saline, Fisher’s exact test, N=8
against both micro-organisms. The effect of
this extract on S. aureus and E. coli was
almost equivalent. Even with high doses, the
aqueous extract (e.g. 1.68 g/kg: P=0.2281
against S. aureus and P=0.1126 against E.
coli) did not show significant antibacterial
activity against both micro-organisms (Table
3). The essential oil with lower doses
inhibited the growth of both micro-organisms
(Table 4). This effect of oil was more
considerable against S. aureus.
Table 2. Effects of intraperitoneal injection of chloroform extract (CE) of N. sativa seed on infected
mice with micro-organisms.
Treatment Dose %Activity
E.coli S. aureus
Normal saline 0.4 ml 0 0
Gentamicin 33 mg/kg 100*** 100***
CE 0.38 mg/kg 75** 75**.
CE 1.5 mg/kg 75** 87.5**
CE 2.6 mg/kg 100*** 100***
** P< 0.01, *** P< 0.001, compared to normal saline, Fisher’s exact test, N=8
DISCUSSION
In this study, the N. sativa seed as an essential
oil (Table 4) , as well as methanol (Table 1)
and chloroform (Table 2) extracts showed
significant antimicrobial activities in vivo.
However, the effect of the aqueous extract
(Table 3) was not significant (e.g. 1.68 g/kg:
P=0.2281 against S. aureus and P=0.1126
against E. coli).

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Table 3. Effects of intraperitoneal injection of aqueous extract (AC) of N. sativa seed on infected
mice with micro-organisms.
Treatment Dose %Activity
E.coli S. aureus
Normal saline 0.1 ml 0 0
Gentamicin 33 mg/kg 100*** 100 ***
AC 0.24 g/kg 2.5 14.2
AC 0.96 g/kg 25 14.2
AC 1.68 g/kg 33.3 37.5
*** P< 0.001, compared with normal saline, Fisher’s exact test, N=8
There are in vitro studies documenting that N.
sativa seed have in vitro antibacterial activity
against pathogens such as S. aureus, E. coli,
Shigella spp.and Vibrio chloerae (Hanafy and
Hatem, 1991; Morsi, 2000; Ferdous et al.,
1992; Rathee et al., 1982). The essential oil of
N. sativa seeds was effective against multiple
drug-resistant (e.g. ampicillin, co-trimoxazole
and tetracycline) isolates of Shigella spp.,
Vibrio chloerae and E. coli in vitro (Ferdous
et al., 1992). Our study replicated these
antibacterial effects against the standard
species of E. coli in vivo. Further, our study
demonstrated for the chloroform and
methanol extracts, as well as the EO of N.
sativa had showed antibacterial effect against
S. aureus. In another study, the antibacterial
activity of N. sativa seed oil was reported
against twenty-one pathogenic bacteria such
as S. aureus and E. coli in vitro.22 This study
beside that confirmed the antibacterial activity
of N. sativa with different extracts it also
showed its effect on micro-organisms in the
body. So now evaluation of black cumin can
be more reliable for doing clinical trails and
pharmacokinetic parameters such as
metabolism or distribution does not prevent
the antibacterial activity of N. sativa.
Filter paper discs impregnated with diethyl
ether extract of N. sativa seeds caused
concentration dependent inhibition of S.
aureus and E. coli (Hanafy and Hatem.,
1991). This effect is similar to the
bacterostatic effect of chloroform and
methanol extracts in this in vivo study.
Thymohydroquinone, a phenolic compound
isolated from the essential oil of N. sativa,
exerted high antimicrobial effect against gram
positive microorganisms (El-Fatatry, 1975).
Another study showed that the most effective
extracts of N. sativa seeds were crude alkaloid
and water extracts but in our study the
aqueous extract showed low antibacterial
activity. This may be related to lower
accessibility of the aqueous extract to the
micro-organism in the in vivo study or low
extraction of antibacterial components into
this extract (Morsi, 2000).
Several compounds such as sterols and
phenolic constituents were found in N. sativa
seed. Thymoquinone or thymohydroquinone
are active components of the seed that have
different pharmacological activities such as

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anti-inflammatory, antioxidant and
antihypertensive effects (Khan, 1999). Higher
solubility of these active constituents in
organic solvents may contribute to the
Table 4. Effects of intraperitoneal injection of Essential oil (EO) of N. sativa seed on infected mice
with micro-organisms.
Treatment Dose %Activity
E.coli S. aureus
Normal saline 0.1 ml 0 0
Gentamicin 33 mg/kg 100*** 100 ***
EO 0.03 g/kg 37.5 50
EO 0.12 g/kg 50 62.5 **
EO 0.21 g/kg 75 ** 87.5 **
EO 0.3 g/kg 100*** 100***
** P< 0.01, *** P< 0.001, compared with normal saline, Fisher’s exact test, N=8
observed efficacy of the chloroform and
methanol extracts and the reason for low
antibacterial activity of the aqueous extract in
our study.
In conclusion, this study and previous reports
show that N. sativa seed has effective
antibacterial activities in both in vitro and in
vivo studies. The result showed that the
essential oil as well as methanol and
chloroform extracts are effective against
Gram-positive (S. aureus) and Gram-negative
(E. coli) bacteria. The isolation and
purification of the active antibacterial
components of plant along with different rout
of administration of plant especially oral way
are recommended. The results of this research
are valuable steps toward doing clinical trials
to use this plant in treating human infection.
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