Black Cumin Seed Essential Oil, as a Potent Analgesic and Antiinflammatory Drug
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PHYTOTHERAPY RESEARCH
Phytother. Res. 18, 195–199 (2004)
Published online in Wiley InterScienceBLACK
(www.interscience.wiley.com).
CUMIN SEED ESSENTIAL DOI: 10.1002/ptr.1390
OIL 195
Black Cumin Seed Essential Oil, as a Potent
Analgesic and Antiinflammatory Drug
Valiollah Hajhashemi1*, Alireza Ghannadi2 and Hadi Jafarabadi1
1
Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences,
Isfahan 81746-73461, Iran
2
Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences,
Isfahan 81746-73461, Iran
The steam-distilled essential oil of Iranian black cumin seed (Nigella sativa L.) was investigated for its
composition and analgesic and antiinflammatory properties. After oil analysis by GC/MS, 20 compounds were
identified in the oil, obtained in 0.4% (v/w) yield. Among them, para-cymene (37.3%) and thymoquinone
(13.7%) were the major components. Acetic acid-induced writhing, formalin and light tail flick tests were used
for assessment of analgesic activity. Antiinflammatory activity was evaluated using carrageenan-induced paw
oedema in rats and croton oil-induced ear oedema in mice. Black cumin seed essential oil (BCSEO) was found
to produce a significant analgesic effect in acetic acid-induced writhing, formalin and light tail flick tests.
Naloxone, an opioid antagonist, could not reverse the analgesic effect observed in the formalin test. Although
oral administration of BCSEO at doses of 100, 200 and 400 µL/kg did not exert a significant antiinflammatory
effect in the carrageenan test, i.p. injection of the same doses significantly ( p < 0.001) inhibited carrageenan-
induced paw oedema. BCSEO at doses of 10 and 20 µL/ear could also reduce croton oil-induced oedema.
It seems that mechanism(s) other than opioid receptors is (are) involved in the analgesic effect of BCSEO
since naloxone could not reverse this effect. Both systemic and local administration of BCSEO showed
antiinflammatory activity. Thymoquinone, as one of the major components of BCSEO, probably has an
important role in these pharmacological effects. Copyright © 2004 John Wiley & Sons, Ltd.
Keywords: black cumin seed; Nigella sativa; Ranunculaceae; essential oil; analgesic; antiinflammatory.
source for the isolation of potential drugs. Evalua-
INTRODUCTION
tion of analgesic and antiinflammatory effects of black
cumin seeds has been the subject of several studies
Black cumin (Nigella sativa L.), Ranunculaceae, is a in recent years. These studies were focused on pure
widely distributed annual herbaceous plant. The seeds thymoquinone (one of the main compounds of N. sativa
of black cumin, commonly known in Iran as ‘Siah fixed and essential oils), fixed oil and aqueous extracts
Daneh’, have been used extensively in foods and of the seeds (Abdel-Fattah et al., 2000; Al-Ghamdi, 2001;
Iranian traditional medicine to treat several disorders El-Dakhakhny et al., 2002; Houghton et al., 1995;
(Zargari, 1990). The seeds have been widely added as Khanna et al., 1993; Mutabagani and El-Mahdy, 1997).
a spice to a variety of Persian foods such as bread, Various pharmacological and biological tests were
yogurt, pickles, sauces and salads for flavouring. They also carried out to investigate different compounds and
are also used in Iranian folk and traditional medicines fractions of black cumin seeds (Agarwal et al., 1979;
for treating some respiratory, gastrointestinal, rheumatic Agel, 1992; Agel and Shaheen, 1996; Boskabady and
and inflammatory disorders (Amin, 1991; Nafisy, 1989; Shirmohammadi, 2002; Burits and Bucar, 2000; El-Tahir
Zargari, 1990). Some Iranian practitioners, such as et al., 1993a, 1993b; Worthen et al., 1998).
Rhazes and Avicenna, were also familiar with this plant The present study was carried out in an attempt to
and mentioned its medicinal uses, such as analgesic and investigate the potential analgesic and antiinflammatory
antipyretic effects in their books, ‘Continens’ and ‘The effects of black cumin seed essential oil, as an out-
Canon’, respectively, (Ebn-e Sina, 1988; Razi, 1990). standing fraction of the plant, in mice and rats using
Black cumin seeds have been reported to contain acetic acid-induced writhing, formalin, light tail flick,
essential oil, fixed oil, flavonoids, saponins, alkaloids carrageenan and croton oil-induced ear oedema tests.
and proteins (Al-Ghamdi, 2001; Burits and Bucar, 2000; In addition, the essential oil constituents identified by
Zargari, 1990). Because the traditional and folkloric GC/MS analysis are described, since some of these con-
uses of black cumin seed are supported by a long his- stituents have been reported to possess antiinflammatory
tory of human experience, this plant may be a valuable and antinociceptive activities.
* Correspondence to: Dr V. Hajhashemi, Department of Pharmacology,
School of Pharmacy and Pharmaceutical Sciences, Isfahan University of MATERIAL AND METHODS
Medical Sciences, Isfahan 81746-73461, Iran.
E-mail: vhajhashemi@hotmail.com
Contract/grant sponsor: Research Council of the Isfahan University of Plant collection and essential oil preparation. N. sativa
Medical Sciences, Isfahan, Iran; Contract/grant number: 80239. L. seeds were obtained from a research farm in Roshan
Copyright © 2004 John Wiley & Sons, Ltd. Phytother.Received
Res. 18,6195–199
February(2004)
2003
Accepted 12 June 2003
Copyright © 2004 John Wiley & Sons, Ltd.196 V. HAJHASHEMI ET AL.
Dasht, Isfahan, Iran, at an altitude of ca. 1400 m in Light tail flick test. Acute nociception was assessed
September 2001, with the aid of a traditional farmer. using a tail flick apparatus (Poya-armaghan, Iran)
The plant material was authenticated by Mr Iraj following the method of D’Amour and Smith (1941).
Mehregan in the Herbarium Department of the Faculty Briefly, each animal was placed in a restrainer,
of Pharmacy, Shiraz University of Medical Sciences, 2 min before treatment, and the baseline reaction
Shiraz, Iran. A voucher specimen, herbarium number time was measured by focusing a beam of light on
HN-1100 has been preserved for reference in the the distal one-third portion of the animal’s tail.
Pharmacy Herbarium of Isfahan University of Medical BCSEO, vehicle or morphine was administered
Sciences, Isfahan, Iran. The air-dried seeds of the plant i.p. immediately after this step and the post drug
were powdered and the volatile fraction was isolated reaction time was measured at 15 min intervals until
by a steam-distillation method for 2 h (Pharmacopoeia 2 h. A 12 s cut-off time was used in order to prevent
Bohemoslovaka IV, 1987). The oil was dried over tissue damage. The MPE% (percent of maximum
anhydrous sodium sulphate and stored in a refrigerator possible analgesic effect) was calculated for each time
(4 °C). interval.
Essential oil analysis. The oil was analysed by GC/MS Carrageenan test. The antiinflammatory activity was
using a Hewlett Packard 6890 mass selective detector evaluated by the carrageenan-induced paw oedema
coupled with a Hewlett Packard 6890 gas chromato- test in the rat (Winter et al., 1962). Male Wistar rats
graph, equipped with a cross-linked 5% PH ME siloxane were briefly anaesthetized with ether and injected
HP-5MS capillary column (30 m × 0.25 mm, film thick- subplantarly into the right hind paw with 0.1 mL of 1%
ness 0.25 µm). Operating conditions were as follows: suspension of carrageenan in isotonic saline. The left
carrier gas, helium with a flow rate of 2 mL/min; hind paw was injected with 0.1 mL saline and used as a
column temperature, 60°–275 °C at 4 °C/min; injector control. The paw volume was measured prior to and
and detector temperatures, 280 °C; volume injected, 4 h after carrageenan administration using a mercury
0.1 µL of the oil; split ratio, 1:50. plethysmograph (Ugo Basil, Italy).
The MS operating parameters were as follows: BCSEO was diluted in 1% Tween 20 and adminis-
ionization potential, 70 ev; ionization current, 2 A; ion tered orally or i.p. 45 or 30 min prior to carrageenan
source temperature, 200 °C; resolution, 1000. injection. The control group received an equivalent
Identification of the components in the oil was based volume of the vehicle. Indomethacin (10 mg/kg) was
on retention indices relative to n-alkanes and computer used as a positive control.
matching with the Wiley 275 L library, as well as by
comparison of the fragmentation patterns of the mass Croton oil-induced ear oedema. The croton oil ear test
spectra with those reported in the literature (Adams, was performed as already described (Carlson et al.,
1995; Sandra and Bicchi, 1987). 1985). A total of 15 µL of an acetone solution con-
taining 75 µg of croton oil was applied to the inner
Animals. Male Swiss mice (25–35 g) and Wistar rats surface of the right ear of each mouse. The left ear
(180–200 g) obtained from the animal house of the remained untreated. Control animals received only the
Faculty of Pharmacy (Isfahan, Iran) were used. They irritant, while in the test groups, in addition to irritant,
were housed in polypropylene cages under standard two doses of 10 and 20 µL of BCSEO were also applied
environmental conditions and had free access to pellet topically. The animals were killed 6 h later and a plug
diet and tap water. For experimentation, six animals (6 mm in diameter) was removed from both the treated
were included in each group. and untreated ear. The difference in weight between
the two plugs was taken as a measure of oedematous
Acetic acid-induced writhing test. Writhing activity response.
was determined by the method of Koster et al. (1959).
Groups of mice (n = 6) received oral different doses Statistical analysis. The results are presented as mean
of BCSEO 1 h prior to an intraperitoneal injection of ± SEM and statistically analysed by one-way ANOVA
1% acetic acid in a volume of 10 mL/kg. The control followed by the Duncan test.
group received vehicle (10 mL/kg of 1% solution of
Tween 20). Indomethacin (10 mg/kg, p.o.) pretreated
animals were used as a positive control.
RESULTS
Formalin test. The test was carried out as described
by Hunskaar and Hole (1987). Mice were injected Analysis of the essential oil
with 20 µL of 2.5% formalin (in 0.9% saline) into the
subplantar space of the right hind paw and the dura- The plant seeds yielded 0.4% (v/w) of a yellowish
tion of paw licking was determined 0–5 min (first phase) essential oil with a fresh flavoured odour and an
and 20–25 min (second phase) after formalin injection. aromatic, slightly bitter taste. Twenty components
Essential oil was given orally or i.p. 45 or 30 min were characterized, representing 97.4% of the total
prior to formalin injection. Control animals received oil components detected, which are listed in Table 1
vehicle (10 mL/kg of 1% solution of Tween 20). with their percentage composition and retention
Morphine (10 mg/kg, i.p.) pretreated animals were indices. Among them, para-cymene (37.3%), thymo-
included in the study for comparison. In some groups quinone (13.7%), linalool (9.9%), alpha-thujene (9.8%)
naloxone (1 mg/kg, i.p.) was injected 15 min before and longifolene (6.4%) were the major components.
BCSEO to assess the role of opioid receptors in anal- Other terpene derivatives formed less than 3.5% of
gesic activity. the total or were present only in trace amounts.
Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 195–199 (2004)BLACK CUMIN SEED ESSENTIAL OIL 197
Table 1. Composition of the essential oil of Iranian black cumin Table 3. Effect of oral and i.p. administration of BCSEO on
seed formalin nociception response in mice (n = 6)
No Compound Retention index Percentage Time spent on licking (s)
(mean ± SEM)
1 alpha-Thujene 924 9.8
First phase Second phase
2 alpha-Pinene 929 3.1
Treatment Dose (0–5 min) (20–25 min)
3 Sabinene 968 2.2
4 beta-Pinene 937 3.4
Control – 43.7 ± 1.6 58.7 ± 9.1
5 alpha-Terpinene 1014 0.8
BCSEO 100 µL/ kg (p.o.) 29.3 ± 2a 20.8 ± 10.5a
6 para-Cymene 1022 37.3
200 µL/ kg (p.o.) 28 ± 3.2a 6 ± 3.5a
7 gamma-Terpinene 1058 2.0
400 µL/ kg (p.o.) 23.7 ± 4.1a 5.5 ± 2.9b
8 Linalool 1091 9.9
100 µL/ kg (i.p.) 24 ± 4.1b 4.2 ± 2.7b
9 cis-Thujone 1096 0.2
200 µL/ kg (i.p.) 19 ± 3.9b 3.2 ± 3.1b
10 Camphor 1138 0.6
400 µL/ kg (i.p.) 17.7 ± 3.5b 0.8 ± 0.8b
11 4-Terpineol 1171 1.0
Naloxone + 1 mg/kg (i.p.) + 12 ± 2.1b 0.7 ± 0.6b
12 alpha-Terpineol 1184 2.2
BCSEO 400 µL/ kg (i.p.)
13 Thymoquinone 1244 13.7
Morphine 10 mg/kg (i.p.) 2.7 ± 1.3b 1 ± 0.5b
14 Bornyl acetate 1282 0.2
15 Thymol 1286 0.4 a
p < 0.01; b p < 0.001 significantly different from control group.
16 Carvacrol 1296 1.6
17 alpha-Longipinene 1348 2.1
18 Citronellyl acetate 1353 0.4
19 Longifolene 1400 6.4 flick test, BCSEO, like morphine, produced antino-
20 Davanone 1593 0.1 ciceptive activity (Table 4). Although the analgesic
effect of BCSEO was less than morphine, it remained
for a longer time.
Table 2. Effect of oral administration of BCSEO on acetic acid-
induced writhing in mice (n = 6)
Antiinflammatory activity
Number of writhings Inhibition
Treatment Dose (mean ± SEM) (%)
Table 5 shows the result of oral and i.p. administration
Vehicle (control) – 47.3 ± 4.9 – of BCSEO on carrageenan-induced paw oedema in rats.
BCSEO 200 µL/ kg 26.3 ± 2.7 43 Orally administered BCSEO at doses of 100, 200 and
400 µL/ kg 24.5 ± 5 48 400 µL/kg produced 24%, 34% and 20% inhibition of
800 µL/ kg 14 ± 6.8a 70 oedema, respectively. However, these effects were not
Indomethacin 10 mg/kg 8.8 ± 2.7a 81 statistically significant. In this model indomethacin,
a standard drug, significantly ( p < 0.001) reduced
a
p < 0.001 compared with control group. carrageenan-induced paw oedema. BCSEO when ad-
ministered i.p. had a better effect, so that the same
doses caused 54%, 74% and 87% inhibition of oedema,
Analgesic activity respectively, and the changes were significant ( p < 0.05
for a dose of 100 µL/kg and p < 0.001 for doses of 200
In the acetic acid-induced writhing test, BCSEO at and 400 µL/kg).
doses of 200, 400 and 800 µL/kg reduced the number The effects of BCSEO on croton oil-induced ear
of abdominal constrictions by 43%, 48% and 70%, oedema are shown in Table 6. BCSEO at doses of 10
respectively. Indomethacin, as a reference drug, pro- and 20 µL/kg significantly reduced ear oedema by 39%
duced 81% reduction of writhes (Table 2). The results and 50%, respectively.
of the formalin test have been summarized in Table 3.
Both oral and i.p. administration of BCSEO, in a dose-
dependent manner, significantly inhibited the formalin-
induced paw licking response. However, the inhibitory DISCUSSION
response on the second phase was more pronounced.
Morphine, as a standard drug, also inhibited both phases The results of this study indicated that black cumin
of formalin-induced pain. Naloxone failed to antagon- seed essential oil has potent analgesic and antiinflam-
ize the antinociceptive effect of BCSEO. In the light tail matory effects. BCSEO showed analgesic activity in all
Table 4. Antinociceptive effect of BCSEO and morphine in light tail flick test in mice expressed
as mean of percent of maximum possible antinociceptive effect (MPE%) of six animals per group
Time after drug administration (min)
Treatment Dose 15 30 45 60 75 90 105 120
Control – 3.6 3.5 3.8 4 3.3 3.8 3.2 3.5
BCSEO 400 µL/kg (i.p.) 6.8 22.4 50.4a 55.9a 57.1a 57.6a 50.8a 51.5a
Morphine 10 mg/kg (i.p.) 32.8a 59.3a 80.9b 87.6b 74.5b 62.1b 45.5a 32.1a
a
p < 0.01; b
p < 0.001 significantly different from control group.
Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 195–199 (2004)198 V. HAJHASHEMI ET AL.
Table 5. Effect of oral and i.p. administration of BCSEO on Table 6. Effect of local administration of BCSEO on croton oil-
carrageenan-induced rat paw oedema in rat (n = 6) induced ear oedema in mice (n = 6)
Increase in paw Ear oedema (mg)
volume (mL) Inhibition Treatment Dose (µL/ear) (mean ± SEM) Inhibition (%)
Treatment Dose (mean ± SEM) (%)
Control – 11.4 ± 0.9 –
Control – 0.41 ± 0.02 – BCSEO 10 7 ± 1.1a 39
BCSEO 100 µL/kg (p.o.) 0.31 ± 0.07 24 20 5.7 ± 0.6b 50
200 µL/kg (p.o.) 0.27 ± 0.05 34
400 µL/kg (p.o.) 0.33 ± 0.05 20 a
p < 0.05; b
p < 0.01 significantly different from control group.
Indomethacin 10 mg/kg (p.o.) 0.11 ± 0.02b 73
Control – 0.39 ± 0.04 –
BCSEO 100 µL/kg (i.p.) 0.18 ± 0.06a 54 oil in different parts of the world. The amount of
200 µL/kg (i.p.) 0.10 ± 0.05b 74 thymoquinone, which was found as a second major
400 µL/kg (i.p.) 0.05 ± 0.05b 87 compound of the essential oil, was lower than that
Indomethacin 10 mg/kg (i.p.) 0.08 ± 0.05b 79
reported in the literature (Burits and Bucar, 2000;
a
p < 0.05; b p < 0.001 significantly different from corresponding
Mozaffari et al., 2000). Many factors can influence the
control groups. essential oil content of plants. These variations are
heredity, age of the plant, climatological environment,
harvesting time, fertilization and irrigation regimens,
animal models used in this study. The formalin test is distillation procedure and isolation method (Gora
considered a valid model for clinical pain (Tjolsen et al., 2002; Omidbaigi, 1997; Reineccius, 1994).
et al., 1992) and in this model, BCSEO effectively Thymoquinone has been partly associated with phar-
inhibited the licking response in both early and late macological findings of the present study (Abdel-Fattah
phases, in a manner similar to morphine. However, et al., 2000; El-Dakhakhny et al., 2002; Houghton et al.,
naloxone, an opioid antagonist failed to reverse the 1995). It has been reported that thymoquinone inhib-
analgesic effect of BCSEO and it seems that mech- ited the generation of thromboxane B2 and leukotriene
anisms other than stimulation of opioid receptors are B4 thus suggesting an inhibitory effect on both the
involved. In a previous report (Abdel Fattah et al., cyclooxygenase and 5-lipooxygenase enzymes (El-
2000), naloxone significantly blocked N. sativa oil and Dakhakhny et al., 2002). Furthermore thymoquinone
thymoquinone-induced antinociception in the early was found by the same authors to inhibit lipid peroxida-
phase of the formalin test, but in our study naloxone tion. The role played by prostaglandins, leukotrienes
had no such effect on the antinociceptive activity of and oxygen radicals in inflammation and pain is well
BCSEO. This difference might be due to different documented (Hardman and Limbird, 2001). Therefore,
chemical composition of fixed oil and essential oil of it seems that at least a part of the analgesic and anti-
the plant. While N. sativa oil is mostly composed of inflammatory effects of BCSEO is due to the presence
fatty acids including linoleic (58%–65%), oleic (22%– of thymoquinone, although it is not so clear whether
24%), palmitic (13%–20%) and stearic acid (Nergiz and this compound is the only contributing component of
Otles, 1993; Saleh Al-Jassir, 1992), none of these com- this plant essential oil or not. Further work is needed
pounds is found in BCSEO. to clarify the role of the other constituents of BCSEO
Since BCSEO showed an antiinflammatory effect, and their exact mechanisms.
it seems that it is more effective in alleviating pain of In conclusion, significant antiinflammatory and anal-
an inflammatory origin. Our results indicate that the gesic activities of black cumin seed essential oil have
antiinflammatory effect of BCSEO is consistent with been found in these models suggesting a rational basis
that reported by Mutabagani and El-Mahdy (1997). for folk and traditional uses of this herb in Iran for
Many of the identified oil compounds were present some inflammatory ailments.
in the essential oils of black cumin seed reported be-
fore (Burits and Bucar, 2000; D’Antuono et al., 2002;
Mozaffari et al., 2000). More than 50% of the essential Acknowledgements
oil components of Iranian N. sativa were due to para- This work was supported by Research Council of the Isfahan
cymene and thymoquinone. There are some variations University of Medical Sciences, Isfahan, Iran (Research project
in the quantitative compositions of N. sativa essential No. 80239).
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