Ethanolic extract of Nigella sativa protects Fe(II) induced lipid peroxidation in rat’s brain, kidney and liver homogenates Waseem Hassan1,3*, Hamsa Noreen1, Shafqat Ullah Khalil1,2, Arshad Hussain2, Shakilla Rehman1, Shagufta Sajjad1, Ata Ur Rahman1 and Joao BT da Rocha3 1Institute of Chemical Sciences, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan 2Food Technology Center, PCSIR Labs Complex, Jamrud Road, Peshawar, Pakistan 3Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Abstract: The study describes the effect of ethanolic extract of Nigella sativa against Fe(II) induced lipid peroxidation. Basal and Fe(II) induced thiobarbituric acid reactive species (TBARS) production was significantly inhibited by the ethanolic extract of Nigella sativa at 25-200µg/ml. Our data revealed that the extract has high DPPH radical scavenging activity at highest tested concentrations. The extract significantly chelated Fe(II) and scavenged hydroxyl (OH●) radical at 25-200µg/ml concentration. The nutritional analysis was performed and carbohydrate, fats, fiber, protein, moisture and ash content were measured in the studied extract. The phytochemical analysis confirmed the presence of alkaloid, carbohydrate & sugar, glycosides, phenolic compounds, flavonoids, protein and amino acid, phytosterols, tannins, gum and mucilage. The extract also showed significant antimicrobial activities against 10 bacterial strains i.e. Salmonella typhi, Bacillus subtilis, Bacillus cereus, Klebsiella pneumonia, Escheria coli, Xanthomonas, Salmonella heidelberg, Staphylococcus aureus, Clostridium and Escheria coli (human) and 5 fungal strains i.e. Aspergillus niger, Entomola, Aspergillus flavus, Alternaria alternata and Penicillium. This study confirms the potential antioxidant and antimicrobial activities of ethanolic extract of Nigella sativa which can be considered not only as a diet supplement but can be used against a variety of free radical induced damage diseases. Keywords: Nigella sativa, lipid peroxidation and antimicrobial activities. INTRODUCTION cellular substrates and ultimately can help in reducing the risk of various diseases. In this regard flavonoids, Reactive oxygen species (ROS) have important phenolic acids, tannins and tocopherols have received physiological functions in living organisms and are attention for their high antioxidant activity (Rice-Evans et actively involved in cellular signal transduction, cell al., 1998) and less toxicity problems which may arise proliferation and even apoptosis (Ames et al., 1993; from the use of synthetic antioxidants (Amarowicz et al., Bland, 1995). The ROS production is counter balanced by 2000; Aruoma et al., 1992). It could be summarized that antioxidant defense systems which include both reducing plants have many Phytochemicals which are potential molecules and various enzymes. The biological situation sources of natural antioxidants and can be useful against a where the imbalance between pro-oxidants and variety of oxidative stress related diseases. antioxidant is disrupted in favor of the former can lead to a process known as oxidative stress (Halliwell & Nigella sativa Linn sometimes known as black seed or Gutteridge, 1990). ROS, which are potent oxidant, can black cumin belongs to botanical family of damage various bio-significant molecules ranging from Ranunculaceae. The seeds of this plant have been used in DNA damage to protein carbonylation and lipid the Southeast Asia and Middle East for a long time peroxidation. The involvement of ROS has been against a variety of diseases like asthma, hypertension, implicated in a variety of pathological manifestations like diabetes, inflammation, arthritis, tumor and cancer, neurodegenerative diseases and aging etc. (Collin, gastrointestinal disturbances (Ali and Blunden, 2003; El- 1999; Floyd, 1998). The area of antioxidant therapy is Din et al., 2006; Ramadan, 2007). The plant is widely exploding in the literature and various classes of chemical used as spice and can be added to hot beverages. It has compounds have been explored for potential antioxidant been reported that the seed of Nigella sativa has over 100 therapy. However, very less success has been achieved different chemical components including various organic with the later because of the complex pharmacological acids, inorganic acids, reducing sugars, alkaloids, and toxicological processes (Amarowicz et al., 2000; flavonoids, sterols, tannins and saponins. The most Aruoma et al., 1992). One of the most important putative pharmacologically active constituents of Nigella strategies could be the use of dietary antioxidant intake; sativa are thymoquinone (30-48%), p-cymene (7-15%), which may inhibit or delay the oxidation of susceptible carvacrol (6-12%), 4-terpineol (2-7%), t-anethole (1-4%) and a sesquiterpene longifolene (1-8%) (Burits and Bucar, *Corresponding author: e-mail: [email protected] Pak. J. Pharm. Sci., Vol.29, No.1, January 2016, pp.231-237 231 Ethanolic extract of Nigella sativa protects Fe(II) induced lipid peroxidation 2000) thymoquinone derivatives like dithymoquinone, saponins, carbohydrate and sugar, glycosides, phenolic thymohydroquinone and thymol (Padhye et al., 2008). compounds, flavonoids, protein and amino acid, phytosterols, tannins and gum and mucilage in the The present work was designed to explore the antioxidant selected plant seeds using standard procedures described activity of the ethanolic extract of Nigella sativa using in- by Sofowora (1993) and Trease and Evans (2000). vitro methods. To get a deeper insight into the therapeutic use of this plant we have determined its protective effect DPPH radical-scavenging against either basal or Fe(II) induced lipid peroxidation in Scavenging of the stable radical, DPPH was assayed in rat’s brain, liver and kidney homogenate. Several other vitro (Hatano et al., 1998). The extract (0-50 mg/ml) was biochemical tests were performed which will give us an added to a 0.5 ml solution of DPPH (0.25 mM in 95% idea to explain the possible mechanism of action of this ethanol). The mixture was shaken and allowed to stand at extract. room temperature for 30 min and the absorbance was measured at 517 nm in a spectrophotometer. Percent MATERIAL AND METHODS inhibition was calculated from the control. Vitamin C was used as a standard compound in the DPPH assay. Chemicals Thiobarbituric acid (TBA), malonaldehyde (MDA), bis- Fe(II) chelation assay dimethyl acetal, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), The ability of the extract to chelate Fe(II) was determined and phenanthroline were purchased from Sigma (St. using a modified method with a slight modification as Louis, MO, USA). Iron (II) sulphate from Reagen (Rio de described by Puntel et al., 2005. Freshly prepared 500 µM Janeiro, RJ, Brazil). All the chemicals used were FeSO4 (150 µl) was added to a reaction mixture analytical grade and were obtained from standard containing 168 µl 0.1 M Tris–HCl (pH 7.4), 218 µl saline, commercial supplier, while the water was glass distilled. and the ethanolic extract of the Nigella sativa (0-25µl). The reaction mixture was incubated for 5 min before the Sample collection and identification addition of 13 µl 0.25% 1, 10-phenanthroline (w/v). The The seeds of Nigella sativa were purchased from local absorbance was subsequently measured at 510 nm in a market of Peshawar in February 2012. The dried plant spectrophotometer. seeds were grinded using pestle and mortar and packed in polythene bags and placed in a dried place for further OH radical scavenging ability extractions. The plant species were identified by experts The ability of the Nigella sativa extract to prevent 2+ in Pakistan Council of Scientific and Industrial Research Fe /H2O2-induced decomposition of deoxyribose was (PCSIR), Peshawar. carried out using the method of Halliwell and Gutteridge (1989). Briefly, freshly prepared ethanol extract (0–100 Ethanolic extract preparation µl) was added to a reaction mixture containing 120 µl 20 The extract of the Nigella sativa was prepared in ethanol; mM deoxyribose, 400µl 0.1 M phosphate buffer, 40 µl 20 briefly, about 1 g of the Nigella sativa was soaked in 100 mM hydrogen peroxide, and 40µl 500 µM FeSO4 and the ml ethanol for 5 min. Thereafter, the mixture was volume were made to 800µl with distilled water. The centrifuged at 2,000 rpm for 10 min. The supernatant was reaction mixture was incubated at 37°C for 30 min and used for the determination of antioxidant activity the reaction was stopped by the addition of 0.5ml of 2.8% (reducing power, Fe(II) chelating ability and OH radical trichloroacetic acid; this was followed by the addition of scavenging ability) and lipid peroxidation bioassay. 0.4ml of 0.6% thiobarbituric acid (TBA) solution. The tubes were subsequently incubated in boiling water for 20 Proximate or Nutritional analysis min. The absorbance was measured at 532 nm in a The proximate analysis (carbohydrate, fats, protein, spectrophotometer. moisture and ash) of Nigella sativa was determined by using the Association of Official Analytical Chemists Animals (AOAC) methods 1990 (AOAC, 1990). Carbohydrate Adult male wistar rats from our own breeding colony value was determined by difference (100- (moisture + ash (250-350 g) were maintained in an air-conditioned room + protein + fat)). The nitrogen content was determined by (22-25°C) under natural lighting conditions, with water Kjeldahl method and multiplied to factor 6.25 to find the and food