Bronchodilator activity of ethyl acetate extract of Nigella sativa

Introduction

The herb N. sativa (black cumin) is commonly known as black seed. It is belonging to the Ranunculacaea family (butter container). The indigenous areas are North Africa, South West Asia and Southern Europe, and also grown in many countries around the world like those within the Center Eastern Mediterranean locale, Iran, India, Syria, Pakistan, Turkey and Southern Europe (1). In Arabic it is known as "Habbat Al-Baraka" or "Al-Habba Al-Sawda" and Kurdish name is "Rashk rashk". As an eastern spice, N. sativa has long been used as a remedy for many acute as well as chronic diseases (2). Therefore, it has gotten to be a family conventional therapeutic plant within the locale (3). Nigella sativa has a long history of traditional folk use in various cultures and has been recognized as a “miracle remedy” for health promotion and disease control(4).

It has been reported that N. sativa seeds possess many therapeutic effects like bronchodilation(5), anti-hypertensive (6), anti-histaminic (7), antioxidant (8,9), anti-inflammatory (10), anti-diabetic (11), immunepotentiation (12) and many other effects. Many reports recommend the use of black seeds for the treatment of different respiratory problems such as asthma and chronic obstructive pulmonary disease (COPD) (13). Several previous studies have shown that N. sativa (70% hydromethanol) exerts its bronchodilatory effect through various mechanisms including; Ca²⁺channel blockade (5,14) and inhibition of histamine release (15).

The present study was conducted to investigate the bronchodilator activity of ethyl acetate extract of N. sativa seeds on isolated rat's trachea and to spot out other attainable underlying mechanism(s) included.

Materials and methods

Plant Extract preparation

The seeds of N. sativa were obtained from local markets in the Duhok city and were verified by herbalists of Department of Forestry, Agriculture College, Duhok University. The seeds were grounded into powder by an electrical grinder. The powder was extracted by maceration method, in which 1000 g of N. sativa powder was soaked in three liters ethyl acetate for 48 hours at room temperature, then Whatman papers were used for filtration. The ethyl acetate yielded a greenish yellow extract coded as NS.EA. The filtrate was concentrated by evaporation under reduced pressure using rotary evaporator (BŰCHI, Switzerland) at a temperature of 40^oC, and the final product was 120 g of NS.EA. Three days of 37^oC and seven days of fresh air are necessary to obtain pure and save extract (without solvent). Ultimately, the extract was entubated and stored at - 20 ^oC until use (16).

Rats

Six male albino rats (Rattus norvegicus) weighting 200 - 300 g (from Department of Biology, College of Science, University of Zakho) were recruited for the current study. Prior to start the experiment, the animals were placed under standard laboratory conditions (22±2ºC and free access to water and libitum with a 12 hrs on light/12 hrs off light) (17). Standard pellets comprising 25.6 % soya, 1.5 % lime stone, 4.4 % oil, 0.63 % salt, 0.062 % choline chloride, 0.158 % methionine, 66.6 % wheat and 0.05 % trace elements were given to the animals (18).

Tracheal Preparation

Rats were euthanized and the neck was incised to remove trachea. After washing the trachea was carefully placed in aerated Kreb's solution. After that, 4 pieces (3 - 5 mm width) of trachea were made from its lower part (18).

Drugs and Chemicals

Drugs and chemicals used in this study are: acetylcholine bromide from McRkin and William ltd; 4-aminopyridine, BaCl2, tetraethylammonium, glibenclamide and methylene blue from Fluka AG, Germany; nifedipine and indomethacin from Medo chemie Ltd, Cyprus and ethyl acetate from BDH England.

Experimental Protocol

Cumulative concentrations of the extract 0.25, 0.37, 0.50, 0.63, 0.75, 0.88 mg/ml were applied to the isolated trachea to construct the dose-response curves (DRCs) as follows; Group I: To investigate the role of K⁺ channels subtypes in the bronchodilation induced by NS.EA extract, the following blockers: 1 mM of 4-AP, 1 mM of BaCl₂, 1 mM of TEA and 10 µM of GLIB were individually pre-treated for 20 minutes on tracheal rings precontracted with ACh (10µM). Group II: To examine the role of Ca²⁺channel in the bronchodilation effect of NS.EA, the rings were preincubated with nifedipine (30µM) for 10 minutes, and with indomethacin (10 μM) for 20 minutes prior to precontraction with ACh (10 µM) and before cumulative application of NS.EA (19). Group III: To demonstrate the role of cyclic guanosine monophosphate (cGMP) in the bronchodilation mediated by NS.EA, the Ach precontracted tracheal rings were preincubated with M.blue (10 µM) for 20 minutes.

Statistical Analysis

The data were converted into a computerized database format. Two-way ANOVA was used for multiple comparisons between the data (for the cell means within the same row) to detect the statistical significance by the use of Graph Pad Prizm program (version 6). Statistical significant was considered when P-value was (P<0.05).

Results

Figure 1 shows the typical dose-response curves (DRCs) of NS.EA in the control tracheal rings and those pretreated with K⁺ channel blockers. The DRC of BaCl₂ shows a highly significant (Pmax for BaCl₂ was significantly decreased from 53.62% (in the control) to 30.31%, and that's for 4-AP and GLIB were non- significantly decreased to 40.14% and 40.13% respectively. However, the E_max of TEA was slightly increased to 60.64% (Table 1).

In figure 2, the DRCs of nifedipine and indomethacin were shifted to right. The indomethacin DRC demonstrates a highly significant (P<0.001) differences at doses 0.63 and 0.75 mg/ml of NS.EA, and (Pmax for nifedipine and indomethacin were significantly lowered to 80.88% and 73.28% respectively (Table 2).

Figure 1: Dose-response curves for the bronchodilatory effect of NS.EA on tracheal rings in absence and presence of 4-AP, GLIB, BaCl₂ and TEA, and pre-contracted with ACh. *= P<0.05.

Table 1: Log IC₅₀ (Log IC₅₀ of CI 95%) and E_max for the bronchodilatory effect of NS.EA on rat's isolated trachea preincubated with GLIB, 4-AP, BaCl₂ and TEA  

Figure 2: Dose-response curves for the bronchodilatory effect of NS.EA on tracheal rings in absence and presence of nifedipine and indomethacin, pre-contracted with ACh. *= P<0.05.

It is revealed in figure 3 that the percentage of relaxation was non-significantly decreased from 55.77% (in the control) to 42.73% (Table 3). Therefore, there is no significant difference between the dose-response curve of the control and that of m. blue.

Table 2: Log IC₅₀ (Log IC₅₀ of 95%) and E_max for the bronchodilatory effect of NS.EA on rat's isolated trachea preincubated with nifedipine and indomethacin

Figure 3: Dose-response curves for the bronchodilatory effect of NS.EA on tracheal rings in absence and presence of methylene blue, and pre-contracted with ACh.  *= P<0.05.

Table 3: Log IC₅₀ (Log IC₅₀ of CI 95%) and E_max for the bronchodilatory effect of NS.EA on rat's isolated trachea preincubated with methylene blue

Discussion

The data obtained from this study revealed that inhibition of COX enzyme produced a potent inhibition of bronchodilation induced by NS.EA extract. This refers to the crucial role of PGI₂ in NS.EA-induced bronchodilation (1) by some active compounds in this plant such as thymoquinone, thymol and carvacrol (20,21). The results of the current study are not comparable due to absence of a corresponding work. However, some earlier in vivo studies revealed that intraperitonial injection of thymoquinone (a N. sativa active compound) in a mouse model had inhibited the protein expression of COX₂, but slightly inhibited COX₁ protein expression (20). Furthermore, thymoquinone component of the essential oil of N. sativa showed an inhibitory effect on both COX and 5-Lipoxygenase of arachidonic acid metabolism in rat's peritoneal leukocytes (22).

On the other side, the results of the current study showed that the soluble guanylate cyclase enzyme has no role in the dilatory effect of the extract. It is impossible to compare the results of the present study, since no similar data on the effect of NS.EA on rat's tracheal soluble guanylate cyclase enzyme are available so far.

In the present work, blocking of L-type Ca²⁺ channels significantly inhibited the extract-induced bronchodilation. This effect suggests presence of sufficient amounts of Ca²⁺ channel blocker components in the NS.EA extract, may be mainly the thymoquinone. Ghayur et al. (23) revealed similar findings when he used thymoquinone. He found that thymoquinone inhibits muscle contraction by interacting with calcium signaling pathways in bronchial smooth muscle of mouse preincubated with verapamil. Another study illustrated that aqueous extract of N. sativa caused relaxation in guinea pig trachea precontracted with CaCl₂ through calcium blocking effect (24).

The effects of NS.EA extract on K⁺ channels revealed that, the extract caused bronchdilation via increasing channel conductance of the K_ir channel. Furthermore, the exploration for the role of K_Ca channels, K_v channels and K_ATP channels in the NS.EA bronchodilation effects were also performed by the current work. It was shown that such K⁺ channels subtypes play no role in this effect.

These results are supported by Keyhanmanesh et al. (25), they suggested the opening effect of aqueous extract of N. sativa on K⁺ channel in guinea pig tracheal rings. However, the channel subtype in trachea in this study was not specified (26). Recently, it was found in a study that flavonoid components (compferol diglucoside) of 20% methanolic fraction of N. sativa induces a bronchdilation effect, although it was lower than that of theophylline. However, the exact mechanism has not been reported, but opening of K⁺ channel (25) and inhibition of muscarinic receptor (27) may be implicated.

We conclude that NS.EA extract causes bronchodilation through the soluble guanylate cyclase, cyclooxygenase, K_ir channel and blockade of Ca²⁺ channel. The various mechanisms of action of bronchodilation indicates presence of a variety of active compounds in NS.EA extract which makes N. sativa versatile in its therapeutic success. N. sativa may be beneficial for controlling asthma. Therefore, in future the plantmay be considered as the object of clinical studies, pharmacological applications and as adjuvants in medicine.

Conclusion

NS.EA extract caused bronchodilation through cyclooxygenase, Kir channel and blockade of Ca₂⁺ channel. The various mechanisms of action of bronchodilation is an indication of presence of a variety of active ingredients in N. sativa which showed the potential of N. sativa in the prevention and/or treatment of respiratory problems.

Acknowledgments

The authors would like to thank the college of Medicine/ University of Duhok and college of Science/ University of Zakho, for providing facilities.

Conflict of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.