Evaluation of The Antioxidant Activity of Ethyl Acetate Fraction of Nigella sativa(L.) Seed Extract On Gastric Mucosal Integrityin Rats.

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Saleh, M.I.A, Isa., A.I, Mabrouk, M. A.,  Mohammed, A., Alhassan, A.W

Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Heba, D.
Animal Science Unit, El-Kasr el-Ain, Cairo University, Cairo, Egypt.
Musa, K.Y.

Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria

All correspondence to: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativaL. is one of the most extensively studied plants both phytochemically and pharmacologically.It is an annual flowering plant, native to Southwest Asia and indigenous to the Mediterranean region, Middle-East, but now widely found in India.In the UnaniTibb system of medicine, N. sativa is regarded as a valuable remedy for a number of diseases including gastric diseases. The present study was carried out to investigate the gastroprotective and antioxidant effect of ETAC fraction of Nigella sativa L. (Family; Ranunculaceae) seed extract on indomethacin-induced gastric ulcer model in adult male albino Wistar rats with the fraction of the extract used at doses of 50, 100 and 200 mg/kg using cimetidine as standard drug. Phytochemical screening of the fraction revealed significance and variations in the presence of flavonoids, alkaloids, tannins, saponins, steroids/triterpenes, glycosides/glucosinolates and free anthraquinones in the fraction, while acute toxicity studies revealed a lethal dose (LD50) above 5000 mg/kg. The extract was administered subcutaneously thirty minutes prior to indomethacin (20 mg/kg). The various parameters studied were malondialdehyde, superoxide dismutase and catalase activities. Pretreatment with the fraction at doses of 50, 100 and 200 mg/kg with the 50 and 100mg/kg doses led to a decrease, though not significant in catalase(CAT) and superoxide dismutase (SOD) activities,while malondialdehyde (MDA) concentration significantly increased (P<0.05)with regard to the extract treated groups. This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased to 200mg/kg. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. In conclusion, the gastroprotective properties of this fraction evaluated may be attributed to its reducing effect on oxidative damage and neutrophil infilteration in tissues due to the presence of phytochemicals like flavonoids, alkaloids and tannins, amongst others, present in the seed extracts with various antioxidant and other biological activities.

Keywords: Nigella sativa, Free Radicals, Antioxidant, NSAID’s,

1.0 INTRODUCTION

Peptic ulcer disease is one of the most common gastrointestinal disorders which causes a high rate of morbidity and at times even mortality particularly in the developing world (Falk, 2001).The pathophysiology of these disorders has focused on an imbalance between aggressive and protective factors in the stomach such as acid-pepsin secretion, mucosal barrier , mucus secretion , blood flow, cellular regeneration, prostaglandins, and epidermal growth factors.Stress, smoking, nutritional deficiencies, and ingestion of non-steroidal anti-inflammatory drugs are all factors which increases the gastric hyperacidity and gastroduodenal ulcer incidences (Lima et al., 2006;Jainu and Devi, 2006).An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage including those of the gastric mucosa that could lead to peptic ulceration (Burkeret al., 2003).Cooperative defence systems that protect the body from free radical damage include the anti-oxidant nutrients and enzymes. The antioxidant enzymes include catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx),and indirectly, glutathione reductase.Their roles as protective enzymes are well known and have been investigated extensively in both in vivo and in vitro model systems.The first three enzymes directly catalyse the transformation of peroxides and superoxides to non- toxic species.Glutathione reductase reduces oxidized glutathione to glutathione; a substrate for GPx.The consequences of oxidative stress are serious,and in many cases are manifested by increased activities of enzymes involved in oxygen detoxification (Kyounget al., 2003).Identification of new anti-oxidants remains a highly active research area, because recently reactive oxygen species generated in the cells of aerobically respiring organisms due to many factors have been implicated in the pathogenesis of many human sufferings like Parkinson’s disease, cancer and gastric diseases (Gutteridge, 1998; Mau et al.,2001; Gulcinet al.,2002b) and anti-oxidants may reduce the risk of various acute and chronic diseases caused by free radicals (Kyounget al.,2003). For the discovery of new leading agents which would be used for the benefit of mankind, we focused our study on the free radical scavenging potentials of Nigella sativa. The plant Nigella sativa has been used for medicinal purposes for centuries, both as a herb when pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being. The seeds are used as carminative, aromatic, stimulant, diuretic, antihelminthic,galactagogue and to increase sweat. They are used as a condiment in curries. A tincture prepared from the seeds is useful in indigestion, loss of appetite, diarrhoea, dropsy, amenorrhoea, dysmenorrhoea and in the treatment of worms and skin eruptions. Externally, the oil is used as an antiseptic. To arrest vomiting, the seeds are roasted and given internally (Gupta et al., 2009).

2.0 MATERIALS AND METHODS:

2.1 Plant material:
Nigella sativadried seeds were obtained during the month of July, 2011 from Sabon-Gari market in Zaria. Botanical identification and authentification were done by Mr. U.A Gallah at the Herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher herbarium specimen (No: 101201) was deposited at the herbarium for future references.

2.2 Extraction and fractionation of the plant material:
Nigella sativa seeds weighing about 2kg were crushed and pounded with pestle and mortar. The powder was extracted with aqueous ethanol (70%) in a Soxhlet Extractor, concentrated using rotaryevaporator at reduced pressure, suspended in ethanol and partitioned with ethyl acetate(ETAC) to obtain the ethyl acetate fraction. The fraction obtained was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue fraction.

2.3 Phytochemical screening of the fraction:
The ethyl acetatefraction was analysed for the presence of
flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins and reducing sugars using standard procedures for analysis (Evans, 2002 and Harborne, 2007).
2.4 Acute toxicity studies:
Lethal Dose (LD50) determination was conducted using the method of Lorke (1983). In the initial phase, male rats were divided into three groups of 3 rats each, making a total of 9 rats, for the ethyl acetate group.The rats were treated with the ethyl acetate fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneously. Animals were observed for 24 hours and the number of death(s) or those that showed neurological signs were recorded. In the second phase, the animals were grouped into 4 groups of one rat each and treated with the ethyl acetate fraction at appropriate doses subcutaneously. The rats were observed for 4 h for deaths or neurological signs, and the final LD50 was calculated as the square root of the highest non-lethal dose in which the animal survived multiplied by the lowest lethal dose in which the animal died.

2.5 Drugs and chemicals/reagents:
Cimetidine (LekPharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories,UK), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA),Phosphate Buffered Saline (PBS) Solution at pH 7.4 (Life Technologies,USA), Heparin (Pfizer Pharmaceuticals, USA), Potassium Phosphate at pH 7.5 (Sigma-Aldrich,USA), Thiobarbituric Acid 25mmol/L (Sigma-Aldrich,USA), Hydrogen Peroxide (H2O2) 500mM/L (OCI Chemical Corporation, USA), Ethylene DiamineTetraacetic Acid (EDTA) (DOW ChemicalCompany,USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA).

2.6 Experimental animals:
A total of fifty eightadult male albino wistar rats were used in this study. The animals were obtained from the Animal House, Faculty of Medicine, El-Kasr el-Ain, Cairo University, Egypt. Their weights ranged from 180 – 240g. They were maintainedunder a similar conditions of humidity, temperature and light/dark cycle respectively and each of the animal was kept in a single individual cage, with wide-meshed galvanized wire bottoms to decrease coprophagy as much as possible.The rats were given access to food and water ad libitum for two weeks to acclimatize, prior to the commencement of the experiment. The rats were treated in accordance to the Principles of Laboratory Animal Care, and experimental protocol was approved by the Animal Ethical Committee in accordance with the guide for the care and use of laboratory animals.At the time of the experiment, all treatments were conducted between 9:00 and 10:00 (GMT+1) h to minimize variations in animal response due to circadian rhythm. The animals were divided into the following groups and subgroups for gastric mucosal damage and gastric secretion studies respectively.

2.7 Experimentaldesign:
Group 1: Normal saline. Five rats received normal saline (1ml/kg/rat subcutaneously (S.C)).
Group 2: Indomethacin-treated.Five rats received indomethacin (20 mg/kg S.C)
Group 3: Cimetidine-treated. Ten rats for the study of the

for the study of the effect of cimetidine, 50 and 100 mg/kg S.C, given 30 mins prior to indomethacin administration (5 rats for each dose).
Group 5: Nigella sativa-treated. Fifteen rats for the study of the effect of ethyl acetate fraction, each at three different doses (50, 100 and 200 mg/kg S.C), when given 30 minutes prior to indomethacin administration (5 rats for each dose).

2.8 Collection of blood samples
Blood samples were collected using heparinized capillary tubes from the retro-orbital plexus of each rat, and kept in EDTA bottles. Immediately, the blood was centrifuged at 1,006 x g at 6oC for 10 minutes, the plasma was obtained and preserved at -20oC until use.

2.9 Collection of tissue samples
From each of the already dissected rats, the liver tissues were removed and placed in tissue sample bottles containing normal saline, ready for sample preparation as a homogenate for oxidative activity assay.

2.10 Determination of anti-oxidant enzyme activities.

2.11 Catalase assay: Determination of CAT activity was carried out according to the method described by Sinha (1972), Passatiet al., (1980) and Achi (1984), with Catalase Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 17.Distilled water 0.9 ml was added to 0.1ml of tissue homogenate and mixed thoroughly. 2.5ml of phosphate buffer was put into a small conical flask; 0.5 ml of tissue homogenate was added; and 2.0ml of H2O2 also added, and a stop watch was started. The reaction mixture thoroughly mixed and the reaction stopped after every 60 seconds for 3 min with dichromate/acetic acid solution. The mixture in the flask was heated in a water bath for 10 minutes at 80oC. Absorbance was read at 570nm. Catalase activity was expressed as unit/mg of protein.

2.12 Superoxide dismutase assay
Determination of SOD activity was carried out according to the method described by Fridovich (1987), with Superoxide DismutaseAssay Kit (Bio-diagnosticR), Egypt. CAT. No. SD 25 21.To 0.1ml of tissue homogenate was added0.9 ml of distilled water to make 1:10 dilution of tissue homogenate. An aliquant mixture of 0.2 ml of the diluted microsome was added to 2.5ml of 0.05M carbonate buffer. The reaction started with the addition of 0.3ml of 0.3 mM adrenaline. The reference mixture contained 2.5ml of 0.05 M carbonate buffer, 0.3ml of 0.3mM adrenaline and 0.2 ml of distilled water. Absorbance was measured at intervals of 30 s up to 150 s at 480nm. SOD activity was expressed as unit/mg protein.

2.13 Determination of Lipid peroxidation (Malondialdehyde):
Lipid peroxidation was evaluated according to the procedure described by Ohkama and Ohishi (1979), as modified by Varshney and Kale (1996), with Lipid Peroxide Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 29.To 1.0 g of stomach tissue, 10ml of 1/150 phosphate buffer (pH 7.0) was added and homogenized. 0.5ml tissue homogenate, 0.5ml saline and 1.0ml of 10% TCA was added.The mixture was centrifuged at 3000rpm for 20 min.0.25 ml of 0.1M TBA was added to the content and mixed well.The mixture was incubated for 1 h at 95oC and allowed to cool down and the supernatant 2.0 ml was measured using spectrophotometer at 532nm. The level of lipid peroxides was expressed as MDA nmol/mg of protein.

2.14 Statistical analysis
All data were expressed as Mean ± S.E.M (standard error of the mean) using SPSS Version 20. Statistical evaluation was done by analysis of variance (ANOVA) followed by post-hoc analysis by Duncan and Scheffe. Values of p<0.05 were considered significant (Microcal Software Inc., Northampton, USA).
RESULTS

Table 1: The Phytochemical Analysis of Ethyl acetate Fraction of Nigella sativa L. seed extract.

Phytochemical Tests Ethyl acetate
Test for flavonoids +++
Test for alkaloids ++
Test for tannins +
Test for saponins –
Test for steroids Trace
Test for glycosides –
Testforanthraquinones +
Test for reducing sugars +
Test for resins –

+ :Presence of the constituents – – Absence of the constituents

Effect of Ethyl acetateFraction on Markers of Oxidative Stress
As shown in table 2, administration of indomethacin 20mg/kg, the values obtained for catalase, superoxide dismutase and malondialdehyde were 4.76 ± 0.16u/mg, 6.21 ± 0.62u/mg and 4.53 ± 2.19nmol/mg of protein respectively. The catalase significantly increased compared to the normal saline control.The cimetidine 50mg/kg and 100mg/kg alone recorded 1.29±0.05u/mg and 5.98±0.30u/mg of catalase activity with the cimetidine 50mg/kg significantly (p<0.05) decreasing compared to indomethacin control, where as the cimetidine 100mg/kg significantly increased compared to the normal saline control. The superoxide dismutase and malondialdehyde in those groups are insignificant. Cimetidine 50mg/kg when administered with indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.59 ± 0.13u/mg, 3.94 ± 0.11u/mg and 4.47 ± 2.52nmol/mg respectively, with the catalase significantly (p<0.05) decreasing, compared to the indomethacin control. In cimetidine 100mg/kg plus indomethacin catalase decreases significantly lower than the indomethacin control with a value of 4.15 ± 0.14u/mg, whereas superoxide dismutase and malondialdehyde were 2.75 ± 0.28u/mg and 3.816 ± 1.45 respectively, and both are insignificant.
In the ethyl acetate fraction 50, 100 and 200mg/kg plus the indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.30 ± 0.15u/mg, 4.16 ± 0.51u/mg and 12.58 ± 1.82nmol/mg respectively for ethyl acetate 50mg/ug, while 1.83 ± 0.08u/mg, 3.56 ± 0.16u/mg and 14.30 ± 1.40nmol/mg were obtained for the ethyl acetate 100mg/kg respectively. For ethyl acetate 200mg/kg, 5.13 ± 0.47u/mg for catalase, 2.72 ± 0.28u/mg for superoxide dismutase and 4.50 ± 1.16nmol/mg for
malondialdehyde. The catalase level activity for ethyl acetate 200mg/kg was significantly higher than the control, ethyl acetate 50mg/kg and 100mg/kg groups.

DISCUSSION AND CONCLUSION
Recent clinical researches shed a light on the NSAID-induced mucosal injuries and the evident roles played by reactive oxygen species (ROS) via the free radical scavenging pro and antioxidant property of several plant species used in folkloric medicine.
Involvement of ROS in pathogenesis of gastric ulceration was first evident from the studies on ischaemiare-oxygenation-induced gastric mucosal injury (Yoshikawa et al., 1989; Yuda, 1993; Perry et al., 1996). The results of these experiments are in line with those previous reports. In this respect, the ETAC-treated groups, both 50 and 100mg/kg showed decreased catalase activity compared to control, decreased SOD activity that was dose-dependent and increased MDA concentration. Indomethacin-induced gastric ulceration was accompanied with a severe oxidative stress in gastric tissue causing damage to key biomolecules such as lipids, which was apparent from the stimulated lipid peroxidation that led to the accumulation of MDA as well as reduction in the gastric activity of CAT. As shown in the present results, the ETAC fraction of N.sativa significantly reversed the indomethacin-induced changes in SOD, CAT and MDA. This significant reduction in lipid peroxide levels along with significant increase in CAT level suggest increased antioxidant activity and prevention of lipid peroxidation by the ETAC fraction of Nigella sativa. Preventive anti-oxidant enzymes such as SOD and CAT are the first line of defence against reactive oxygen species (ROS) (Yogenderet al., 2007). Flavonoids and other phenolic compounds of plant origin have been reported as scavengers and inhibitors of lipid peroxidation (Formica and Regelson,1995) and are known to have the capacity to sequester endogenously and exogenously produced ROS and free radicals, thereby inhibiting mucosal damage (Repetto and Llesuy, 2002). This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. The cimetidine treated groups were insignificant.
It has been shown that compounds isolated from Nigella sativa have appreciable free scavenging properties (Buris and Bucar, 2000). These might be attributed to various mechanisms among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxide, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging ability (Diplock, 1997; O’Ktayet al., 2003). The ethyl acetate fraction exhibited free-radical scavenger activity as well as primary antioxidant that react with free radicals, which might possibly limit free radical damage, occurring in the body. ETAC fraction of Artemesiaapiacea was found to possess strong antioxidant activity and the major metabolite isolated was flavonoid cacticin (Lee et al., 2002). Similarly, the extracts from Culcitumreflexumhave been reported to possess photo-protective and anti-oxidant activities and their major compounds were flavonoids (Aquino et al., 2002). In line with the present findings, many researchers have indeed demonstrated the in vivo and in vitro anti-oxidant and free radical scavenging activities of flavonoids (Cao et al., 1997; Rice-Evans et al., 1997; Brown et al., 1998; Plumb et al., 1999; Pietta, 2000). Matsuda et al. (2003) reported that flavonoids are the major secondary metabolites class with several descriptions of antiulcer, antioxidant and other gastroprotective properties. Essential oils which are mixtures of wide variety of volatile terpene hydrocarbons (aliphatic and cyclic) were reported in N.sativa(Magiatiset al.,2002). Several terpenoid-containing extracts from plants commonly used in non-orthodox medicine to treat different gastric illnesses have been reported (Arrietaet al., 2003; Schmerda-Hirschmannet al.,2005; Morikawaet al.,2006). According to Esteveset al. (2005), the essential oil from Cascariasylvestris, which contains sesquiterpinebicy-clogermacrene as the major component, displays mucosal cytoprotectiveproperty. Baccharisdra-cunculifolia essential oil also showed a similar activity (Klopellet al., 2007; Lemosetal., 2007).

CONCLUSION
These studies suggested that the ETAC fraction of Nigella sativa protects against free radicals and possessed appreciable free radical scavenging property by promoting oxidant/antioxidant balance,hence it might play a role in combating gastric ailments like peptic ulcers.

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