Nigella sativa fixed and essential oil modulates glutathione redox enzymes in potassium bromate induced oxidative stress

Background Nigella sativa is an important component of several traditional herbal preparations in various countries. It finds its applications in improving overall health and boosting immunity. The current study evaluated the role of fixed and essential oil of Nigella sativa against potassium bromate induced oxidative stress with special reference to modulation of glutathione redox enzymes and myeloperoxidase. Methods Animals; 30 rats (Sprague Dawley) were divided in three groups and oxidative stress was induced using mild dose of potassium bromate. The groups were on their respective diets (iso-caloric diets for a period of 56 days) i.e. control and two experimental diets containing N. sativa fixed (4 %) and essential (0.3 %) oils. The activities of enzymes involved in glutathione redox system and myeloperoxidase (MPO) were analyzed. Results The experimental diets modulated the activities of enzymes i.e. glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) positively. Indices of antioxidant status like tocopherols and glutathione were in linear relationship with that of GPx, GR and GST (P < 0.01). MPO activities were in negative correlation with GST (P < 0.01) but positive correlation with some other parameters. Conclusions Our results indicated that both Nigella sativa fixed and essential oil are effective in improving the antioxidant indices against potassium bromate induced oxidative stress.


Background
Since ages, communities relied on traditional medicines to prevent and cure various types of dietary health disorders. Scientists have realized now plant-based dietary interventions as the core heart epicenter of complimentary and alternative medicines. As the industrialization spun, the trends shifted towards reliance on pharmaceuticals. However, there are several societies across the globe who are more battened towards dietary therapeutic interventions [1,2].
Nigella sativa L. (black cumin or black seeds) is good source of nutritionally essential components. Various cultures and civilizations utilize its seeds as herbal medicine to treat and prevent number of diseases [3]. Its health enhancing potentials are due to active ingredients (thymoquinone, carvacrol, thymol, cymene, t-anethole and 4-terpineol) that are mainly concentrated in fixed or essential oil [4]. Aforementioned bioactive components along with some others constituents possess notable antioxidant activity. N. sativa and its essential ingredient like thymoquinone are effective free radical scavengers in lipid peroxidation. Furthermore, they can also act as immune boosters and several research investigations have supported their anti-inflammatory and immunomodulatory effects [5].
The production of free radicals is an integral part of body metabolism but imbalance results is oxidative stress. The excessive lipid peroxidation results in destruction of cellular membranes that could leads to cell death and degenerative disorders [6]. The modulation of lipid peroxidation through natural body defense mechanism involving hepatic enzymes is of considerable importance. Previously, scientists elaborated that the N. sativa essential oil tends to normalize the level of lipid peroxidase (LPX), lactate dehydrogenase (LDH), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) enzymes [7]. Some other researchers outlined the importance of N. sativa as antioxidant that could be useful in pathological conditions related to free radicals. However, the combination and in vivo role of oxidative stress and antioxidants is still a matter of conjecture. The antioxidant potential seems inversely linked with oxidative damage, however, the interactions between antioxidant potential of N. sativa in oxidative stress conditions needs research interventions conducted comprehensively. In this context, we have tried to study the role of Nigella sativa fixed and essential oils (NSFO & NSEO) in improving the antioxidant status and attempted to explore their mechanisms and molecular targets. For the purpose, we measured the oxidative damage due to oxidative stress and determined the antioxidant potential of the body through measuring total antioxidant capacity (TAC), tocopherols, and glutathione contents. Later, we attempted to explore the mechanism and measured the glutathione redx enzymes expressions and myeleperoxidase activity.

Methods
Nigella sativa seeds were procured from Barani Agricultural Research Institute, Chakwal (Voucher specimen No. Chk.Pk-926) and preserved in the same institute for future reference. The seeds were further identified by Dr. Qasim Ali, Department of Botany, University of Agriculture, Faisalabad. Chemical Reagents (analytical & HPLC grade) and standards were purchased from Sigma-Aldrich Tokyo, Japan and Merck KGaA, Darmstadt, Germany. The present research was conducted following the instructions of "Animal Care Committee, NIFSAT-Faisalabad Pakistan" (NIFSAT/ACC/07/16) and research plan was submitted to National Institute of Health (NIH), Islamabad for further approval of research. They provided infectious free Sprague dawley rats as per requirements of modified proposed research.

Extraction of Nigella sativa fixed and essential oils
Following the standard procedures, the seeds of Nigella sativa were slurred with hexane in 1:6 ratio (Soxtech apparatus and rotary evaporator) to extract the fixed oil. Nigella sativa essential oil was extracted using locally assembled hydro-distillation apparatus. Earlier to this manuscript, the authors published the results pertaining to the nutritional composition of NSFO and NSEO and safety assessment in Food and Chemical Toxicology [8].

Housing of rats
The National Institute of Health (NIH), Islamabad provided infectious free 30 Sprague Dawley rats that were further divided into three groups of ten rats each. The animals were maintained according to standard guidelines of Animal Institute of Nutrition (AIN), USA i.e. temperature 23 ± 2°C, relative humidity 55 ± 5 %, and 12-h light-dark cycle. In the first week, the feed of the rats was basal diet in order to acclimatize them to new environment. After the acclimatization period, rats received their respective experimental diets for a period of 8 weeks (56 days) ( Table 1).
In order to induce oxidative stress in rats, the peritoneal injection of potassium bromate @ 45 mg/Kg body weight dissolved in 0.05 M citrate buffer (pH 4.5) was employed. The analytical procedures carried out include feed & water intake (measured daily) and body weight (weekly basis). At 28 & 56 days of feeding trials, five rats from each group were decapitated for blood collection through neck and cardiac puncture [9]. The collected blood samples were analyzed for further assays and details are mentioned herein.

Indices of oxidative damage
Indicators of lipid peroxidation were estimated including MDA level [10], total antioxidant capacity [11] and conjugated dienes according to protocol described by Corongiu and Milla [12]. For total antioxidant capacity, we added reagent-1 (xylenol orange, NaCl and glycerol) in 35 μL of collected serum and than added 11 μL of reagent-2 (ferrous ion and o-dianisidine, H 2 SO 4 solution) and readings were taken at 560 nm and 800 nm. Briefly, MDA was measured with a colorimetric method using tetraethoxypropane as standard and units followed were nmol/mL. For the determination of conjugated dienes, serum was homogenized and centrifuged at 3000 g. The spectra obtained (232-247 nm) using hexane as standard solution was further used to estimate conjugated dienes and values were expressed in nmol/g of lipids.

Indices of antioxidant status
Glutathione contents were determined following the protocols described by Beutler [13]. In the protein free supernatant, the colored product of GSH + DTNB was measured at 412 nm and expressed as nmol/mg protein. Level of serum α-tocopherol and γ-tocopherol were also measured following the procedures described by Xu and Godber [14] through HPLC. Briefly, the oil samples were slurred in hexane as a first step. A normal phase HPLC column (250 mm × 4.6 mm, 5.0 μm particle size) and mobile phase consisting of isooctane & ethyl acetate (96:4 v/v) was used for the purpose. Total run time and flow rate were 30 min and 1.0 mL/min, respectively. The detector was set at 290 nm excitation wavelength and 400 nm emission wavelengths. The column temperature was 35°C. Similarly, the individual standards of isomers of tocopherols were run using the same pattern and curves were obtained and used for calibration in order to determine the amounts of tocopherols in Nigella sativa fixed oil.

Superoxide dismutase and catalase enzymes assays
The antioxidant enzymes assays include the estimation of Superoxide dismutase (SOD) and catalase. The measurement of SOD activity was based on the reaction between ability of enzyme to inhibit cytochrome 'c' oxidation [15] and activity was expressed in IU/mg protein. The decomposition of hydrogen peroxide and measurement of its by-products were mainly used for the estimation of catalase (CAT) activity. The units remained the same as IU/mg protein and 1 IU was equivalent to 1 μmol H 2 O 2 consumed per mg protein per minute [16].

Glutathione redox enzymes assays
Glutathione transferase (GST) action was measure using the commercial kits provided by Bioassay Internationals. The reaction include the rate of formation of conjugate between GSH and 1-chloro-2,4-dinitrobenzene [13] and measurement unit used IU/mg protein. The 1 IU is equivalent to 1 μmol of conjugate formed/min/mg protein.
Glutathione peroxidase (GPx) activity was estimated using tertiary butyl hydroperoxide (tbHP) as substrate [17] and the activity was expressed in IU/mg protein. The 1 IU is equivalent to one nmol of NADPH oxidized/mg protein in one minute. Glutathione reductase (GR) activity was assayed by estimating the oxidation of NADPH. Glutathione reductase (GR) activity was assayed at 37°C and 340 nm by estimating the oxidation of NADPH. Hepatic enzymes like GST, GPx and GR was determined as described by Paglia and Valentine [18].

Myeloperoxidase, xanthine oxidase, and nitric oxide
In this study, we used spectrophotometer to estimate the activity of tissue associated myeloperoxidase (MPO) following the procedures laid down by Hillegas et al. [19]. For the purpose, single unit of enzyme activity (IU/mg of proteins) was defined as the amount of the MPO present that caused a change in absorbance measured at 460 nm during the reaction time of three minutes. The indices like xanthine oxidase and nitric oxide are also of significance importance as far as immunopotentiating properties are concerned. We measured the xanthine oxidase using the using diagnostic kits from Cayman Chemicals.

Results
The onset of 21 st century witnessed the coinage of new terms like functional, nutraceutical and pharma foods. The awareness among the masses regarding the diethealth linkages improved significantly that further led scientists to conduct research studies. The present research intervention focused on exploring the role of Nigella sativa fixed oil (NSFO) and essential oil (NSEO) to improve antioxidant status and modulation of glutathione redox enzymes. The results pertaining to the different parameters presented herein.

Indices of oxidative damage
Diets affected total antioxidant capacity (TAC), serum MDA and conjugated dienes levels significantly, whilst non-momentous impact of study intervals was observed (

Indices of antioxidant status
It is apparent from

Superoxide dismutase and catalase enzymes assays
The antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) were significantly affected as a function of diets, study intervals and their interaction (  (Table 4).

Glutathione redox enzymes assays
The experimental diets affected the enzyme expressions of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione transferase (GST) significantly (Table 4)

Discussion
The diet and health linkages are well defined and many research studies carried out in animal and humans provided evidences that dietary modules are best strategy in preventing many ailments e.g. cardiovascular disorders and diabetes mellitus [4]. Nigella sativa is famous in Asian countries owing to its potential role in prevention of different ailments. In this regard, Sultan et al. [8] carried out the safety assessment of Nigella sativa fixed and essential oil (NSFO & NSEO). In the present research, further efforts were made to study the effects of NSFO and NSEO on hepatic enzymes, antioxidant status and immunopotentiating potential of Nigella sativa in oxidative stressed Sprague dawley rats. The protocols followed in the present research were based on the studies conducted by scientists [20,21].
In the present study, we fed the groups of rats on experimental diets containing N. sativa fixed and essential oils along with control for a period of 8 weeks. The addition of N. sativa fixed and essential oil increased feed and water intakes as compared to control group that certainly be due to the remedial action of experimental diets thus resulting in restoration of normal metabolism and body homeostasis [22][23][24]. Oxidation and its complexities results in uncontrolled free radicals production that become lethal leading to oxidative stress. In this regard, these reactive oxygen species (ROS) overcomes the antioxidant capacity of the target cells and tissues. In stress-induced conditions, supplementation of antioxidants may evoke certain protective roles in the body against various health discrepancies [25]. In the instant study, total antioxidant capacity (TAC) of the body decreased significantly in control group, while values for serum MDA and conjugated dienes levels increased indicating more oxidative damage. However, diets containing N. sativa fixed and essential oils assuaged the damage by improving total antioxidant capacity by 1.5 and 2.0 folds, respectively. Moreover, serum MDA level decreased significantly by 20.58 and 51.62 % in fixed and essential oils groups, respectively. The conjugated dienes levels also decreased significantly i.e. 15.98 and 34.35 %, respectively. However, levels of serum MDA and conjugated dienes were higher as compared to normal rats owing to injection of potassium bromate used for induction of oxidative stress [8]. Total antioxidant capacity (TAC) is primarily dependent on antioxidants defence system capabilities and secondly on antioxidants ingested as a part of the diet. Moreover, it also relies on total free radical trapping potential of the body in both normal and oxidative stress conditions. The decreased TAC in oxidative stressed rats can be due to depletion of antioxidants present in the body and increased free radical production. Correlation also provided the evidence that MDA and conjugated dienes were negatively associated with TAC. The investigations conducted by Al-Othman et al. [26] proved that black cumin supplementations hold potential to improve the antioxidant capacity of the body. Antioxidant status i.e. glutathione, αand γ-tocopherols were improved consequently with experimental diets. Level of glutathione decreased in control by 25.23 %, while increased by 4.30 and 21.82 % in N. sativa fixed and essential oils groups, respectively. The restoration of glutathione to near normal ranges is hallmark of the present study. Glutathione along with tocopherols were in linear linkage with antioxidant capacity, while inverse association with that of oxidative damage. These findings further validated our stance that supplementation of diets rich in tocopherols and antioxidant ameliorate the adverse consequences of oxidative stress. Production of free radicals results in lipid peroxidation thus resulting in extensive oxidative damage as indicated by high level of MDA & conjugated dienes. These indicators were modulated in groups of rats consuming fixed and essential oil based diets. According to Alenzi et al. [27], the oils of black cumin and thymoquinone served a protective mechanism against toxicity induced by cyclophosphamide and prevent incidence of necrosis. Formerly, Meral and Kanter [28] observed reduced level of MDA, improved antioxidant capacity and restoration of glutathione because of black cumin supplementation that strengthened the present results. Lipid peroxides and associated moieties are products of lipid peroxidation that are harmful in progression of diseases such as atherosclerosis and brain damage. However, experimental diets attenuated the damage that certainly be attributed to their rich nutritional profile. In this context, El-Desoky et al. [29] suggested the potential role of tocopherols and nutraceuticals in scavenging free radicals and ameliorating their toxic effects for normal functionality of the body. In another research investigation, Sánchez-Reus et al. [30] reported that oxido-reduction or GSH/GSSG ratio is important in controlling the antioxidant potential of the body. In the present study, NSFO and NSEO modulated the hepatic enzymes activity. The aforementioned results indicated the involvement of complex mechanism in which specialized processes involving glutathione and tocopherols scavenge the oxidants like MDA, lipid peroxides, hydro-peroxides and conjugated dienes. The expressions of hepatic enzymes enhance the antioxidant capacity in the groups of rats fed on experimental diets. In this context, glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione transferase (GST) are important biological catalyst to control peroxides and free radicals production through glutathione cyclic transformation [31].
Role of enzymes in amelioration of oxidative stress is indispensable. Casalino et al. [32] supported the present findings that antioxidant supplementation could avert the hepotocellular damage as a consequences of oxidative stress. The enhanced level of these metabolites triggers cascade of events that results in malfunctioning of the body and even in severe cases death may be the possible penalty. The supplementations of diets with appropriate antioxidant or formulation containing bioactive compounds are useful in reversing the sequential distortion due to free radicals. Some other studies also suggested that active ingredients present in N. sativa fixed and essential oils protect the body from nephrotoxicity and hepatotoxicity [4]. N. sativa fixed and essential oils ameliorate oxidative stress and maintain body homeostasis by regulating several hematological and serological attributes. The present exploration concluded that N. sativa fixed and essential oils have potential to improve the antioxidant status of the body. The diet supplemented with the N. sativa oils with special reference to essential oil can act as safeguard against oxidative stress and allied disorders. Future perspectives include testing their effectiveness as nutraceuticals in maladies characterized by oxidative stress like arthritis and osteoporosis.

Conclusion
Conclusive approach drawn from this efficacy study is, Nigella sativa fixed and essential oils hold potential to improve the antioxidant status and decrease antioxidant damage significantly in potassium bromate induced oxidative stressed rats. The oxidative stress and ROS induced negative changes in enzyme expressions but experimental diets modulated hepatic enzymes and immune system including GR, Gpx, GST, MPO, and xanthine oxidase positively. However, there is need to conduct further trials in other types of animal modeling to validate the findings.