Indian Journal of Experimental Biology Vol. 43, July 2005, pp. 635-639

Effect of aqueous extract from ·Nigella sativa L. on guinea pig isolated heart

Mohammad Naser Shafei, Mohammad Hossein Boskabady* & Heydar Parsaee

Dep~rtment of Physiology, Ghaem Medical Centre, Mashhad University of Medical Sciences, Mashhad, 91735 , Iran

Received 12 July 2004; revised 27 March 2005

A potent inhibitory' effect of aqueous extract from N. sativa on calcium channel of guinea pig heart was found comparable and even greater than that of diltazem. The results may also indicate an opening effect for the plant on ' potassium channel of isolated heart.

Keywords: Nigella sativa, Aqueous extract, Calcium channel blocker, Isolated heart, Guinea pig

Nigella sativa L. (Ranunculaceae) is a grassy plant with greenish-blue flowers and small black seeds, which grows in temperate and cold climate areas. The seeds of N. sativa contain thymoquinone, monotropens such as p-cymene and a-pinene' , nigellidine2

, nigellimine3 and a saponin4. Several therapeutic effects including those on

digestive disorders, gynaecology, and anti-asthma and dyspnea have been described for the seeds of N. sativa in ancient Iranian medical books5. N. sativa has long been known for its medical use as an antispasmodic5

, especially against gastrointestinal disorders or respiratory ailments, in many countries.

There is evidence of relaxant effects of volatile oil from this plant on different smooth muscles including rabbit aorta6

, rabbit jejunum?, and isolated tracheal muscles of guinea pigs8

. Mahfous and El-Dakhakhny9

reported that the volatile oil from N. sativa protected guinea pigs against histamine-induced bronchospasm, but it did not affect histamine H, receptors in isolated tissue. However, in an in vivo study, increasing respiratory rate and intra tracheal pressure of guinea pigs due to iv administration of volatile oil from N. sativa has been demonstrated lO

• Boskabady et al. have shown, a relaxant effect on guinea pig tracheal chains " , the anticholinergic and histamine H, receptor blocking effects Or! isolated guinea pig tracheal chains '2, and an inhibitory effect on calcium channels of guinea pig tracheal chains'3.

In the present study, the inhibitory effect of aqueous extract from N. sativa on isolated guinea pig hearts has been examined.

*Correspondent author Fax: +98 511 413579 E-mail : m-boskabady@mums.ac.ir.mhboskabady@hotmail.com

Materials and Methods Plant and extracts-No sativa was collected from

Torbat Heydarieh (northeast Iran) during the spring of 2002, and dried at room temperature in absence of sunlight. Botanists in the herbarium of Ferdowsi University of Mashhad identified the plant; and the specimen number of the plant is 293-0303-1. The aqueous extract was prepared as follows: chopped, dried plant (50 g) was extracted with 300 ml-distilled water by suxhelat apparatus. The solvent of the extract was then removed under reduced pressure and distilled water was added so that the plant ingredient concentration in the final extracts was IOg/1 00 ml.

Preparation of the isolated hearts-Dunkin Hartley guinea pigs of either sex, weighing 400-500 g, were provided by Razi Institute, Mashhad, Iran. Guinea pigs were killed by a blow on the neck; the hearts were rapidly isolated and washed in ice-cold saline. The coronary circulation was perfused through aorta on a modified Langendorff apparatus at a constant perfusion pressure of 70 mm Hg'4. The hearts were perfused with K-H buffer solution (37°C, pH 7.4, saturated with 95 % O2 and 5 % CO2) . The K-H buffer solution contained the following (in mmo\/\): NaCl118, NaHC03 25, KCI4.7, KH2P04 1.2, MgS04 1.2, CaCh 2.5, and glucose 11 equilibrated with 95% O2 + 5% CO2 at 37°C. All the hearts were first perfused with K-H solution for 20-30 min for stabilization in the Langendorff apparatus and then the effects of extracts from N. sativa and diItiazem were studied.

Protocol of experiments-The effects of four different concentrations of aqueous extract from N. sativa (0.5, 1, 2 and 5 mg % from the extract) and diItiazem (0.1, 1, 10 and 100 ~M) on heart rate and heart contractility compared to baseline values were

636 INDIAN J EXP BIOL, JULY 2005

examined. For studying the influences of different concentrations of each solution, each concentration of the solutions was given as one-minute intracoronary infusion and the heart rate and contractility were recorded in the last 30 sec. During the experiments each heart served as its own control before injection of each solution. In absence of pharmacological intervention, both, heart rate and contractility were reproducible l5

. The effects of different solutions were tested with two different experimental designs as follows:

1. Perfused heart with ordinary Krebs solution (group 1 experiments, n=8).

2. Perfused heart with calcium free Krebs solution (group 2 experiments, n=7).

The effects of the extract and diltiazem in two groups of experiments were examined in two different series of animal hearts. The effects of aqueous extract and diltiazem in each heart were performed randomly with a 30 min resting period of the heart between examining the effect of each two solution while the heart was perfused with ordinary Krebs. In all experiments, the heart rate (RR) and contractility were recorded on a kymograph (ET8 G-Boulitt, Paris) and were measured after fixation. The local Animal Research Committee of Mashhad University of Medical Sciences approved the experimental procedures used in the present study.

Statistical analysis-The data of the heart rate and contractility were expressed as mean±SE. The data of heart rate and contractility obtained in presence of different concentrations of extract, diltiazem, and baseline were compared using ANOV A test in each

group. The effect of each concentration of aqueous extract and diltiazem between two groups was compared using unpaired t test. The effect of aqueous extract and diltiazem were related to the concentrations of the solutions using least square regression. Significance was accepted at P<0.05 .

Results Effecte of aqueous extract on heart rate- In group

1 experiments last three concentration of diltiazem (1, 10 and 100 ~ and aqueous extract (1, 2 and 5 mg%) significantly reduced heart rate of guinea pigs compared to baseline values (Table 1). In group 2 experiments, higher concentration of diltiazem (100 JlM) and three higher concentrations (1, 2 and 5 mg%) of the aqueous extract significantly reduced heart rate of guinea pigs compared to baseline values (Table 1).

Effect of aqueous extract on heart contractilitr-In group 1 experiments, 10 and 100 ~ concentrations of diltiazem, 1, 2 and 5 mg % concentrations of aqueous extract significantly reduced heart contractility of guinea pigs compared to baseline values (Table 2). In group 2 experiments 100 !lM concentration of diltiazem, and 3 higher concentrations (l, 2 and 5 mg %) of aqueous extract significantly reduced heart contractility of guinea pigs compared to baseline values (Table 2).

Differences between the effect of diltiazem and aqueous extract-In group 1, the effect of higher concentration (5 mg%) of aqueous extract on both heart rate and contractility was significantly higher than diltiazem (Fig. 1 a and c).

In group 2, the effects of two higher concentrations (2 and 5 mg%) of aqueous extract on heart rate and heart contractility were significantly greater than that of diltiazem (Fig. 1 b and d).

Table 1- Effect of four different concentrations of aqueous extract from N. sativa and diltiazem on heart rate (beats/min) of isolated guinea pig' s hearts

[Values are mean ±SE]

Experimental design Groupl P Group 2 P P (n=8) (vs B) (n=7) (vs B) (vs G.I)

Baseline 269.75±12.36 158.66±i .96 <0.001 Diltiazem 0.1 239.00±15.18 NS 152.66±9.65 NS <0.001 (lAM) 1.0 196.00±12.28 </).01 149.33±11.01 NS NS

10 140.75±12.47 <0.00 1 128.66±1O.04 NS NS 100 70.75±4.58 <0.001 1 16.66±9.54 <0.05 NS

Baseline 272.64±11.80 164.52±8.84 <0.001 Aqueous 0.5 228.75±15.28 NS 147.66±9.33 NS <0.001 extract 1.0 183.75±15.46 <0.05 123.66±11.l6 <0.05 <0.01 (mg %) 2.0 106.00±16.96 <0.01 81.33±19.56 <0.01 NS

5.0 32.00±7.26 <0.001 39.33±13.29 <0.001 NS

Group I: experiments on isolated guinea pig heart in presence of ordinary Krebs solution. Group 2: experiments on isolated guinea pig heart in presence of calcium free Krebs solution. St. Dif: Statistical difference, NS: non significant difference, B: baseline, D: diltiazem.

SHAFEl et al.: EFFECT OF NIGELLA SATIVA ON GUINEA PIG ISOLATED HEART

Table 2-The effect of four different concentrations of aqueous extract from N. sativa and diltiazem on contractility of isolated of guinea pig' s hearts in two groups of experiments

Experimental design

Baseline

DiItiazem (J.lM)

Baseline Aqueous extract (mg%)

For abbreviations see Table I.

280 260 240

(].) . 220 ro 200 ... 180 1::: 160 co 140 (].)

::r: 120 100

80 60 40 20

2 .5

-0> 2.0

~ 1.5

13 ~ C 1.0 o U 0.5

0 .0

0

o

0.1 1.0 10 100

0.5 1.0 2.0 5.0

Group I

2.2I±O.20 2.06±O.23 l.S4±0.22 0.95±O.19 0.44±O.14

2.2I±O.20 2.02±0.21 1.62±O.17 1.31±0.IS 0.2S±O.OS

2

2

3

3

P (vs B)

NS NS <0.01 <0.001

NS <0.05 <0.01 <0.001

(a)

4

1.5

1.0

0.5

(c) .. 0.1

4

Group 2

1.20±O.09 1.l1±0.OS 0.97±0.091 0.91±0.07 0.77±0.06S

1.20±O.09 1.02±0.10 0.SO±O.07 0.65±0.09 0.34±0.1O

160

140

120

100

80

60

40

20

0

o

Concentration

P P (vs B) (vsG. I)

<0. 01 NS <0.01 NS <0.05 NS NS <0.01 NS

<0.01 NS <0.01 <0.05 <0.01 <0.01 NS <0.001 NS

NS

(b)

2 3

Diltiazem Aqueous extract

NS

(d)

2 3 4

4

637

Fig. l-Concentration response curves of aqueous extract from N. sativa, and diltiazem, on heart rate (beats/min) and contractility (g) of isolated guinea pigs in group I (in presence of ordinary Krebs solution, n=S). (a and c) and group 2 experiments (i n the presence of calcium free Krebs solution. n=7), (b and d). Tested concentration for extract were 0.5,1,2 and 5mg% and for diltiazem 0.1. 1. 10 and lOOJ.lM. Statistical differences between the effect of extract with that of diltiazem; NS: non-significant difference. p values; *<0.05, **<0.01, ***<0.001.

Differences ill the effects of extract between two groups of experiments-The baseline heart rate prior to addition of diltiazem and aqueous extract in group 2 were significantly lower than those of group 1 (P<O.OOI forall cases, Table 1). In addition the heart rate obtained in presence of only first concentration of diltiazem (0.1 1lM), and two lower concentrations of

aqueous extract (0.5 and 1 mg%) were significantly lower than those of group 1 (Table 1). Baseline heart contractility obtained prior to addition of diltiazem and aqueous extract in group 2 were also significantly lower than those of group 1 (Table 2). The heart contractility in presence of two lower concentrations of diltiazem (0.1 and 1.0 ~M) and aqueous extract (0.5

638 INDIAN J EXP BIOL, JULY 2005

and 1.0 mg%) in group 2 were also significantly lower than those of group 1 (Table 2).

Relationship between concentration and the effect of aqueous extract and diltiazem-There was significant negative correlation between both heart rate and heart contractility with different concentrations of diltiazem and aqueous extract (Table 3).

Discussion The results of the present study showed a

concentration dependent decrease in both heart rate and heart contractility due to aqueous extract from N. sativa in presence of ordinary Krebs solution (group 1 experiments). The effect of aqueous extract was very similar to that of diltiazem.

In group 1, three higher concentrations of diltiazem (1.0, 1O.and 100 JlM) and aqueous extract (1, 2 and 5 mg%) of N. sativa significantly reduced heart rate. In this group, three higher concentrations of aqueous extract (1. 2 and 5 mg%) and two higher concentrations of diltiazem 10 and 100 JlM) significantly inhibited the heart contractility. The results of group 1 indicated a potent and concentration dependent inhibitory effect of aqueous extract from N. sativa on heart rate and contractility. The effect of aqueous extract on heart rate and contractility were very similar to those of diltiazem. These similarities may suggest an inhibitory effect of the extract from N. sativa on calcium channels of isolated heart.

To explore the effect of extracts of this plant on calcium channels more precisely, their effects on heart rate and contractility of isolated heart were re­examined in presence of calcium free Krebs solution in group 2 experiments. In this group, three higher concentrations of aqueous extract (1, 2 and 5 mg%) reduced heart rate and contractility. The effects of aqueous extract on heart contractility were similar to those on heart rate in group 1, but highest concentration of diltiazem (100 JlM) also significantly inhibited the heart contractility.

Table 3-Correlation between the effects of aqueous extract from N. sativa on heart rate and contractility of isolated guinea pig heart with concentrations in two groups of experiments

Experimental Agueous extract Diltiazem groups r P value r P value Group I HR -0.845 <0.001 - 0.713 <0.001

Cont - 0.466 <0.001 - 0.356 <0.001

Group 2 HR - 0.743 <0.001 - 0.263 <0.01 Cont - 0.542 <0.001 - 0.342 E<O.OOI

The effect of highest concentration of aqueous extract (5 mg%) in group! reduced both heart rate and contractility to the same level as those of group 2

. indicating complete inhibition of calcium channel.

These findings indicated that aqueous extract of N. sativa almost completely inhibited calcium channels of isolated guinea pig heart. The significant negati ve correlation between concentrations of different solutions and their effect on heart rate and heart contractility strongly supported concentration dependent effects of aqueous extract and diltiazem.

There are three groups of calcium channel blocker drugs including: (i) dihidropyridins such as nifidipine and nicardipine which act mainly on smooth muscle (ii) fenylalkylarnins such as verapamil which mainly act on heart muscle, and. (iii) benzodiazepines such as diltiazem that act on smooth muscle16

• The results of the present study may indicate that effect of aqueous extract from N. sativa is more similar to the fenylalkylarnin group. In fact. the result of the present study concerning depressant effect of diltiazem on heart rate is supported by study of Gerhard et at. 17

The diltiazem concentration used on the present study was also very similar to those of above study l7.

However, the minute conce~trations of aqueous extract from N. sativa caused similar effect to the concentration of diltiazem used in the present study. These findings may indicate that the aqueous extract of N. sativa has a very potent inhibitory effect on calcium channels and/or opening effect on potassium channels of guinea pig isolated heart.

Comparison of the effect of aqueous extract and diltiazem also showed that the effect of aqueous extract on both heart rate and contractility of group 1 were very similar to those of diltiazem. These findings suggest that aqueous extract inhibited calcium channels of isolated heart in a very similar manner to diltiazem. The slopes of concentration response curves in presence of extract were similar to those obtained in the presence of diltiazem for both heart rate anJ contractility in group 1. In group 2, the effect of aqueous extract on heart rate was similar but higher than that of diltiazem. However, the effect of highest concentration of aqueous extract on contractility in group 2 was higher than that of diltiazem. The slopes of concentration response curves in presence of extract were different from those obtained in presence of diltiazem for both heart rate and contractility. The greater effect of aqueous extract from N. sativa on heart rate in group 2 and differences in the slope of concentration response

SHAFEI et al. : EFFECf OF NIGELLA SATIVA ON GUINEA PIG ISOLATED HEART 639

curves between extract and diltiazem may indicate an additional effect for aqueous extracts other than calcium channel inhibitory effect. This additional effect could be a potassium channel opening effect for extract, because positive inotropic effect of potassium channel blocker has been demonstrated l8

. In fact, in an earlier study a potassium channel opening effect for extract of N. sativa has been suggestedl9

.

The other mechanisms such as cholinergic stimulatory and/or adrenergic inhibitory effects of the plant could be responsible for the observed effect of aqueous extract on isolated heart. However, Boskabady et at. showed anticholinergic I I and ~­adrenergic stimulatory20 effects for the plant. The concentration of calcium, potassium and sodium in extract also may account for its heart effects. Therefore, the concentrations of these ions in the extract were examined. The concentration of calcium in extract was in normal range (11 mg %) but potassium concentration of the extract was high (18.8 mmol/l) and that of sodium was low (29.6 mmolll) in extract with 109/1 00 ml. The high concentration of potassium could account for reduction in contractility and low concentration of sodium could be responsible for both reduction in contractility and heart rate. However, the extract was diluted 2000 times in highest experimental concentration (5 mg%). Therefore, the concentration of calcium, potassium and sodium would not affect heart rate and contractility in extract concentrations used in the present study. However, the exact mechanism of this plant on heart rate and contractility should be clarified in further studies.

In conclusion, the results of the present study suggest a possible potent inhibitory effect of aqueous extract from N. sativa on calcium channels of isolated guinea pig heart. However, the action of aqueous extract on potassium channel may differ partially from that of diltiazem. Acknowledgement

This study was financially supported by the Vice Presidency of Research of Mashhad University of Medical Sciences. Refrences

El-Dakhakhny M, Studies on chemical constitution of Egyptian Nigella sativa L. seeds. II. The essential oil, Planta Medica, 11 (1963) 465.

2 Atta U R & Malik S 0 , Nigellidine, a new indazol alkaloid from seeds of Nigella sativa, J Res Inst, 36 (1995) 1993.

3 Atta U R & Malik S 0, Nigellimine N-oxide, a new isoguinoline alkaloid from the seeds of Nigella sativa, Hetrocycles, 23 (1985) 953.

4 Ansari A K & Sadiy HAS, Structural studies ori a saponin isolated from the seeds of Nigella sativa, Phyto Chem, 27 (1998) 377.

5 Ave-Sina, Law in medicine, (Ministry of Guidance publication, Tehran, Iran) 1990,313.

6 Agel M B, The relaxing effect of volatile oil of Nigella sativa seed on vascular smooth muscle, Jordan Ser Bul, 1 a (1992) 91.

7 Agel M B, Effects of Nigella sativa seeds on intestinal smooth muscle, 1m J Pharmacogn, 31 (1993) 55.

8 Reiter M & Brandt W, Relaxant effects on tracheal and ileal smooth muscles of the guinea- pig, Arzneim ForschlDru8 Res, 35 (1985) 408.

9 Mahfouz M & El-Dakhakhny M, Chemical and pharmacological properties of the new anti-asthmatic drug, nigellone, Egypt Phaml Bull, 42 (1960) 411 .

10 El-Tahir K E H, Ashour MMS & Al-Harbi MM, The respiratory effect of the volatile oil of the Black seed (Nigella sativa) in guinea pig: elucidation of the mechanism(s) of action, Genfharmacol, 24 (1993) 1115.

11 Boskabady M H & Shahabi M, Bronchodilatory and anticholinergic effects of Nigella Sativa on isolated guinea­pig tracheal chains, Iran J Med Sci, 22 (1997) 127.

12 Boskabady M H & Shiravi N, Inhibitory effect of Nigella Sativa on histamine (HI) receptors of isolated guinea pig tracheal chains, Eur Respir J, 16 (2000) 461.

13 Boskabady M H & Shirmohammadi B, Inhibitory effect of Nigella sativa on calcium channels of isolated guinea pig tracheal chains, Arch I ran Med, 5 (2002) 103.

14 Chlopicki S, Kozlovski V I & Gryglewski R J, No-dependent vasodilation induced by nebivolol in coronary circulation is not mediated by beta-adrenoceptors or by 5 HTIA-receptors. J Physiol Pharmacol, 53 (2002) 615.

15 Chlopicki S, Kozlovski V I & R J Geygewski, Clonidine­induced coronary vasodilation in isolated guinea pig heart is not mediated by endothelial a2 adrenoceptors, Physio Pharmacology, 54 (2003) 511.

16 Hurwitz L, Partridge D & Lean JK, Calcium channels: properties, junctions, regulation alld clinical relevallce , l SI edition, (CRC Press, Florida) 1991,77.

17 Gerhard S, Karin K, Ulrike S & Nobuaki M, Effects of semotiadil, a novel Ca2+channel antagonist, on the electrical activity of Langendorff-perfused guinea pig hearts in comparison with diltiazem, amlodipine and nifedipine, ElIr J Pharmacol, 286 (1995) 71.

18 Kocis 1, Gru-chae A & Petrusewicz J, Gender differences in effects of pinacidil but not diazoxide on heart automatism in the isolated guinea pig right atria, J Phanllacoi, 55 (2003) 419.

19 Boskabady M H, Shirmohammadi B, Jandaghi P & Kiani S, Possible mechanisms for relaxant effect of agueous and macerate extracts from Nigella sativa on tracheal chains of guinea pig, BMC Pharmacol, 4 (2004) 3.

20 Boskabady M H, Kiani S, Jandaghi P, Stimulatory effect of Nigella sativa on Bz-adronceptors of guinea pig tracheal chains, Med J I R lrall, 18 (2004) 153.