Effect of Nigella sativa L. seed extract on cisplatin-induced delay in gastric emptying in Sprague-Dawley rats

Abstract The aim of this study was focused on investigating the possible protective effect of Nigella sativa L. seed extract against cisplatin-induced delay in gastric emptying, in a rat model. Twenty-five male Sprague-Dawley rats were divided into five equal groups as follows: Group I or control group, Group II (cisplatin 10 mg/kg, i.p at day 5), Group III (N. sativa L. 250 mg/kg for 5 days + cisplatin 10 mg/kg, i.p on day 5), Group IV (N. sativa L. 500 mg/kg for 5 days + cisplatin 10 mg/kg, i.p on day 5) and Group V (ondansetron 3 mg/kg/day, per os + cisplatin 10 mg/kg, i.p on day 5). Phenol red meal was adopted to estimate gastric emptying in different groups of the rats. Gastric emptying was significantly increased (p < 0.01) in N. sativa L. seed extract-pretreated rats (Group III and Group IV) when compared to cisplatin treatment alone (Group II). However, ondansetron produced significantly (p < 0.01) better reversal than N. sativa L. seed extract.


Introduction
Cisplatin [cis-diamminedichloroplatinum (II)], one of the most widely used anticancer drugs in the world (Matysiak & Gustaw-Rothenberg 2009), is indicated for various malignancies including head, neck, ovarian and testicular cancers (Muggia 2009). However, use of antineoplastic drugs is usually associated with the development of numerous side effects, including anorexia, weight loss and gastrointestinal symptoms such as nausea/emesis, abdominal discomfort and diarrhoea (Di Fiore & Van Cutsem 2009). Approximately, 28-36% of patients receiving an initial dose (50-100 mg/kg) of cisplatin develop acute renal failure (Schrier 2002) and acute gastrointestinal tract disorders ( Takeda et al. 2008). These gastrointestinal disorders by cisplatin are due to its delaying effect on gastric emptying. Gastric emptying refers to a natural process of evacuation of food from stomach into the duodenum. Delayed gastric emptying most commonly gives rise to abdominal discomfort after meals, causing nausea and vomiting. These cisplatin-induced side effects in the gastrointestinal tract may be treated by metoclopramide (Roos et al. 1981;Ullah et al. 2015), 5HT3 receptor antagonists (Kishibayashi et al. 1993;Yanai et al. 2013) and antioxidants (Sharma & Gupta 1997).
The seed of Nigella sativa L. (NS) known as black cumin is a promising medicinal plant component reported to have potent antioxidant effects, and many therapeutic effects of the NS seed have been documented, including antiviral (Barakat et al. 2013), anti-schistosomiasis (el Shenawy et al. 2008, anti-inflammatory (Marsik et al. 2005;Majdalawieh & Fayyad 2015), antimicrobial (Mehta et al. 2009;Sarwar & Latif 2015), immunomodulatory activities (Tekeoglu et al. 2007;Majdalawieh & Fayyad 2015), anti-tumour properties (Linjawi et al. 2015) and inhibit polymorphonuclear leukocytes functions (Kacema & Meraihi 2009). Also, it is been found to attenuate toxic side effects like nephrotoxicity, hepatoxicity induced by several chemotherapeutic agents (Yaman & Balikci 2010;Kushwah et al. 2014). However, it is not known if N. sativa L. seed extract can alter the cisplatin effect on gastric emptying. Therefore, in the present study, the effect of N. sativa L. seed extracts was investigated against cisplatin-induced gastroparesis in rats.

Results and discussion
In the present study in rats, intraperitoneal administration of cisplatin (10 mg/kg) significantly (p = 0.003) inhibited the gastric emptying of a methylcellulose meal to 59.92 ± 0.49% as compared to 84.99 ± 2.78% in saline-treated rats (Group I) ( Figure S1). These results are consistent with the finding that cisplatin causes a delay in gastric emptying in rats (Sharma & Gupta 1998;Cabezos et al. 2010). It is currently thought that cisplatin causes a delay of gastric emptying through a mechanism similar to that by which cisplatin induces emesis. excess of 5-hydroxytryptamine (5-HT) released by cisplatin from enterochromaffin cells in the gastrointestinal tract may stimulate the peripheral 5-HT receptors on the vagal afferent fibers and enteric nerves. This stimulation has been suggested to cause relaxation of the stomach possibly leading to a delay of gastric emptying ( Rudd & Andrews 2005;Stern et al. 2011). Moreover, cisplatin-induced free radical generation in the intestine (Sangeetha et al. 1990) may lead to subsequent release of 5HT (Matsuki et al. 1993;Cui et al. 2016) which may act on peripheral 5HT3 to relax the stomach and lead to the delay in gastric emptying. Similar to the findings of earlier researchers (Kishibayashi et al. 1993;Sharma & Gupta 1998;Ando & Takagi 2011;Yanai et al. 2013), we also observed the reversal of the inhibition by ondansetron.
N. sativa L. seed extract and its active ingredient, thymoquinone, have been characterised as antioxidant and anti-inflammatory agents in different experimental settings (Salem 2005;Inci et al. 2013). In light of these considerations, effect of N. sativa L. seed extract on cisplatin-induced delay in gastric emptying was investigated in rats. Five days pretreatment with N. sativa L. seeds extract at the doses of 250 and 500 mg/kg, per os (Group III and Group IV, respectively) ameliorated the cisplatin-induced delay of gastric emptying by 1.3 and 1.27 folds, respectively, as compared to cisplatin alone (Group II) (N. sativa: 77.9 ± 2.24% at 250 mg/kg, 76.53 ± 1.88% at 500 mg/kg; cisplatin alone: 59.92 ± 0.49%). The reversal of delayed gastric emptying was statistically significant (p = 0.004) at both doses ( Figure S1). To the best of our knowledge, this is the first study to show the protective role of N. sativa L. extract against the inhibitory action of cisplatin on gastric emptying. Therefore, this novel finding can be beneficial in management of the nausea, emesis and accompanying gastrointestinal symptoms such as abdominal discomfort and anorexia induced by cisplatin therapy. The present investigation found that there was no significant difference (p = 0.020) in the effect on gastric emptying between two doses of N. sativa L. extract, 250 and 500 mg/ kg. The possible cause, for no further improvement in gastric emptying by increasing dose to 500 mg/kg may be due the presence of other ingredients in the N. sativa L. seeds that may produce a counter-acting effect at this higher dose. An alternate explanation could be that 250 mg/kg was simply enough to reach the maximum effect, may be thymoquinone is unable to induce a higher effect.
The beneficial effect of N. sativa L. seed extract could be explained by its free radical scavenging property (Inci et al. 2013) and nitric oxide synthetase regulation action (Magdy et al. 2012). Thymoquinone and its metabolite, considered to be the key constituent that affords protection due to its antioxidant and anti-inflammatory activity (Marsik et al. 2005;Inci et al. 2013), its antioxidant property might be due to its scavenger ability for superoxide, hydroxyl radical and singlet molecular oxygen (Kruk et al. 2000;Nagi & Mansour 2000;Badary et al. 2003). Also cisplatin has been found to cause inhibition of neuronal nitric oxide synthetase (nNOS) activation leading to distention of stomach (Jarve & Aggarwal 1997). N. sativa L. seed extract or thymoquinone is thought to regulate nitric oxide production by improving the reduced neuronal nitric oxide by increasing nNOS activity (Magdy et al. 2012) and by suppressing nitric oxide over production due to inhibition of expression of the inducible nitric oxide synthase (iNOS) and iNOS mRNA expression (el-Mahmoudy et al. 2002). Further experimental work needs to be done to establish its mechanisms of its action.

Conclusion
In conclusion, N. sativa L. seed extract inhibits the cisplatin-induced delay in gastric emptying in rats and therefore this novel finding can be beneficial in management of the nausea, emesis and accompanying gastrointestinal symptoms such as abdominal discomfort and anorexia induced by cancer therapy. Further research is required to determine the exact mechanism by which N. sativa L. seeds reduce the cisplatin-induced delay in gastric emptying.

Supplementary material
Supplementary material relating to this article with respect to experimental section is available online alongside Figure S1.