Nigella sativa ameliorates oxidative stress induced adverse effects in rodent modeling studies: Indices of serum chemistry and hematology

Herbs and herbal products have gained immense recognition owing to their health promoting potential. Moreover, scientific innovation and technological advancement provided evidences to support the pivotal linkages between bioactive components and human health (Bjelakovic et al., 2014). Resultantly, functional and nutraceutical foods have emerged as the leading trend in the present decade (Douaire & Norton, 2013). Rich phytochemistry of plants is an important arena of research in the domain of nutrition and pharmacy. Many traditional plants are in use since long to prevent and cure various maladies (Butt & Sultan, 2013). Amongst, Nigella sativa (black seed) is quite common in the South Asia, Middle East, Sub-Saharan regions along with some parts of South-East Asia e.g. Malaysia and Indonesia. Compositionally, its bioactive components chiefly reside in its fixed or essential oil e.g. healthy fatty acids, dihomolionolenic acid, tocopherols, phytosterols, and alkaloids like nigellicine, nigellidine & nigellimine (Cheikh-Rouhou et al., 2007). Furthermore, volatile fractions (essential oil) of N. sativa seeds are rich in antioxidants like thymoquinone, ρ-cymene, carvacrol, t-anethole, and 4-terpineol; possess varied antioxidant activity (Nickavar et al., 2003).


Introduction
Herbs and herbal products have gained immense recognition owing to their health promoting potential. Moreover, scientific innovation and technological advancement provided evidences to support the pivotal linkages between bioactive components and human health (Bjelakovic et al., 2014). Resultantly, functional and nutraceutical foods have emerged as the leading trend in the present decade (Douaire & Norton, 2013). Rich phytochemistry of plants is an important arena of research in the domain of nutrition and pharmacy. Many traditional plants are in use since long to prevent and cure various maladies (Butt & Sultan, 2013). Amongst, Nigella sativa (black seed) is quite common in the South Asia, Middle East, Sub-Saharan regions along with some parts of South-East Asia e.g. Malaysia and Indonesia. Compositionally, its bioactive components chiefly reside in its fixed or essential oil e.g. healthy fatty acids, dihomolionolenic acid, tocopherols, phytosterols, and alkaloids like nigellicine, nigellidine & nigellimine (Cheikh-Rouhou et al., 2007). Furthermore, volatile fractions (essential oil) of N. sativa seeds are rich in antioxidants like thymoquinone, ρ-cymene, carvacrol, t-anethole, and 4-terpineol; possess varied antioxidant activity (Nickavar et al., 2003).
Across the globe, scientists have attempted to explore aforementioned rich phytochemistry of N. sativa to reduce the disease burden. Due to presence of antioxidants, it can be effective in scavenging free radicals and ameliorating the oxidative stress (Sultan et al., 2014). The oxidative stress and its complications could lead to several health disparities including degenerative and neurological disorders. In the last few years, many scientists across the globe investigated the therapeutic potential of black cumin and its bioactive components (Kanter et al., 2009).
The results from such studies indicated its role in prevention of diabetes mellitus, cardiovascular, and respiratory disorders. It also retrains the free radicals produced in the body that results in adverse consequences to health (Falak & Jamil, 2013).
Previously, safety assessment and toxicological evaluation was conducted (Sultan et al., 2009) in normal Sprague Dawley rats. However, the metabolism differs in normal and diseased conditions, as the metabolism is complex to understand completely. In instant research work, we attempted to elucidate the toxicological aspects related to N. sativa fixed and essential oils in oxidative stress conditions. The findings of the present research are helpful in warranting antioxidant potential of N. sativa fixed oil (NSFO) and essential oil (NCEO) in improving antioxidant status and decreasing the oxidative damage in potassium bromate oxidative stress.

Materials and methods
The Barani Agricultural Research Institute (BARI), Chakwal provided us Nigella sativa seeds. Sigma-Aldrich Tokyo, Japan and Merck KGaA, Darmstadt, Germany provided us chemical Reagents (analytical & HPLC grade).

Extraction of Nigella sativa fixed and essential oils
In the present study, we extracted N. sativa fixed oil using Soxtech apparatus. For the purpose, we slurred N. sativa seeds with solvent (hexane was used and its ratio was optimized i.e. 1:6). Like fixed oil, we extracted the volatile fraction using hydro-distillation apparatus that was assembled locally in Faisalabad in collaboration with Azeem Scientific Company, Faisalabad, Pakistan.

Induction of oxidative stress and evaluation of test substances
The test substances were N. sativa fixed oil @ 4.0% and N. sativa essential oil @ 0.30%. The oxidative stress was induced in rats with the peritoneal injection of potassium bromate @ 45 mg/Kg body weight in 0.05M citrate buffer (pH 4.5). The rats were further fed on oxidized corn oil with POV (120 meq/g) to yield chronic oxidative stress.

Test animals and their housing
National Institute of Health (NIH), Islamabad provided infectious free 30 Sprague Dawley rats (Age: 6-7 weeks, weight: 130 ± 10 g). The animals were further divided into three groups of ten rats each and provided them basal-diet (AIN-76A) during the first week to get them acclimatize. The oxidative stress was induced as mentioned in the previous section and three groups received one control & two experimental diets, respectively, for a period of 8 weeks. The compositions of experimental diets include casein (20.0%), corn starch (55.0%), cellulose (10%), corn oil (10.0%), Mineral mixture (4.0%), and vitamin mixture (1.0%). Nigella sativa fixed and essential oils were added in the experimental diets @ 4.0% and 0.30%. In case of fixed oil group, corn oil was reduced accordingly and added in the amounts of 6.0%. In the whole study, we made efforts to strictly abide the standard guidelines of Animal Institute of Nutrition (AIN), USA. Drinking water was provided in bottles, however, feed and water intake were checked daily, while weight of each rat was documented weekly throughout the experimental period. Thrice in the study (0,m 28, and 56 days), five rats from each treatment were sacrificed to collect blood for further analysis (Uchida et al., 2001).

Blood lipid profile
For the estimation of blood lipid profile, we used the collected blood samples from each group of rats and parameters estimated were cholesterol, HDL, LDL, and triglycerides. Serum cholesterol level was determined using CHOD-PAP method, HDL Cholesterol Precipitant method for HDL, liquid triglycerides (GPO-PAP) method for total triglycerides (Annoni et al., 1982) and difference method was used to calculate low density lipoproteins (LDL) following the procedure of (Annoni et al., 1982;Assmann, 1979), (Stockbridge et al., 1989), (McNamara et al., 1990).

Serum glucose and insulin levels
For the purpose, GOD-PAP method was followed to determine the glucose concentration of individual rats (Thomas & Labor, 1992). In comparison, ELISA immunoassay technique was employed to observe the values of insulin (Keilacker et al., 1987).

Serum Biochemistry
Total proteins, albumins, globulin and A/G ratio are all indicators for serum proteins profile and these were also measured using commercial kiets. Additionally, levels of electrolytes were also assessed to check the efficacy of N. sativa fixed and essential oil.

Hematological aspects
Blood samples collected were analyzed for complete blood profile like total red blood cells count, hemoglobin, and hematocrit. Platelets count and erythrocytes sedimentation rates (ESR) were also estimated. Total white blood cells (WBC), neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles were also determined.

Statistical analysis
Regarding the statistical data analysis, values presented in Tables are means ± standard deviation. In order to check the level of significance, we applied analysis of variance (ANOVA) technique and used two factor factorial design and means for diets, study intervals and their interactions were further compared through Duncan's multiple range test (DMRt) following the outlines of (Steel et al., 1997).

Results
The onset of 21 st century witnessed mass awareness regarding the diet-health linkages. The purpose of the study was to exploit the rich phytochemistry of N. sativa oils against oxidative stress. In order to induce oxidative stress, we used intra-peritoneal injection of potassium bromate @ 45 mg/kg body weight (Sultan et al., 2012). The results pertaining to the different parameters are discussed herein.

Feed & water intake and body weight
It is obvious from the means that groups of oxidative stressed rats fed on diets containing N. sativa fixed and essential oils showed higher feed intakes as compared to significantly lower intake in control group (Figure 1). Likewise, the maximum intake was recorded in NSEO group that was statistically at par NSFO but significantly different than the minimum intake control/placebo group. Likewise, means depicting body weight showed a persistent decline control as compared to increasing trend for body weight experimental diets. Figure 1 represents the feed & water intake and body weight. It depicted the decreasing tendency in feed intake and body weight during study in placebo. However, the experimental diets significantly improved these parameters.

Serum lipid profiles and hypoglycemic perspectives
The lipid profile includes cholesterol, HDL, LDL, and triglycerides. It is evident from the means ( Table 1) that N. sativa fixed and essential oils based diets were statistically at par in lowering cholesterol significantly with recorded values of 99.82 ± 5.33 and 102.56 ± 3.13 mg/dL, respectively as compared to 130.82 ± 11.84 mg/dL in control group. In control group, LDL contents increased significantly from 47.44 ± 3.04 to 85.64 ± 5.33 mg/dL whereas in fixed and essential oils groups, significant decrease for this trait from 44.82 ± 2.89 to 31.35 ± 1.84 mg/dL and 45.77±1.81 to 39.24±1.68 mg/dL, respectively. Data showing the means for HDL contents indicated the non-significant variations due to diets ranged from 37.77 ± 0.65 to 39.32 ± 0.90 mg/dL. Serum triglycerides increased in oxidative stressed rats fed on control diet from 108.69 ± 6.97 to 128.35 ± 7.98 mg/dL during study. In contrary, fixed and essential oils groups showed significant decrease from 108.24 ± 6.97 to 86.42 ± 5.08 and 104.43 ± 4.12 to 90.52 ± 3.87 mg/dL, respectively. The groups of rats fed on essential oil exhibited significant decrease in blood glucose level from 113.11 ± 4.47 to 102.40 ± 4.38 mg/dL during 56 days study duration. Likewise, N. sativa fixed oil decreased the same trait from 114.13 ± 2.91 to 105.29±3.57 mg/dL. However, control group showed marked increase in blood glucose from 116.22 ± 6.05 to 138.14 ± 3.58 mg/dL. N. sativa essential oil group showed maximum insulin secretions (46.06 ± 1.50 μU/mL) followed by fixed oil group (44.78 ± 0.90 μU/mL), while minimum level (40.30 ± 1.62 μU/mL) recorded in control. The diets containing fixed and essential oils enhanced the insulin secretions distinctly, however, the same trait dropped significantly in control group.

Indices of serum biochemistry
The means (Table 2) elucidated that diets affected globulin and A/G ratio significantly, while remained non-significant for total protein and albumin contents. However, with the passage of time ratios for albumin contents increased significantly from 3.54 ± 0.08 at start to 4.20 ± 0.20 mg/dL at 56 days of study. The minimum value for globulin contents were recorded in control, while the maximum in NSEO. Diets depicted significant impression on A/G ratio in serum of oxidative stressed rats however, maximum A/G ratio 1.44 ± 0.147 mg/dL was recorded in D1 (placebo), followed by 1.20 ± 0.043 mg/dL in D2 (N. sativa fixed oil) and least 1.16 ± 0.067 mg/dL was recorded in D3 (N. sativa essential oil). The levels of potassium were found to be 4.44 ± 0.10, 4.31 ± 0.07 and 5.40 ± 0.60 mEq/L in D1 (placebo), D2 (N. sativa fixed oil) and D3 (N. sativa essential oil), However, maximum levels of bicarbonates 27.48 ± 2.91 mEq/L in NSEO, while the least level 24.77 ± 2.66 mEq/L was established in NSFO group.

Red blood cell indices
Hematological parameters included red blood and white blood cell indices. The results regarding hematology (Table 3) explicated that red blood cell varied non-significantly as a function of experimental diets. However, significant variations in the mean values were established for hematocrit % i.e. 38.68 ± 2.11, 40.94 ± 1.62 and 45.67 ± 1.37 in control, NSFO, and NSEO group, respectively. The data pertaining to hemoglobin contents clearly indicated the minimum contents in control as compared to 14.95 ± 0.12 and 14.90 ± 0.25 mg/dL in NSFO, NSEO, respectively. The maximum platelet count of 4.65 ± 0.32 K/µL was recorded in N. sativa essential oil followed by 4.47 ± 0.35K/µL in N. sativa fixed oil group and the least plate count was recorded (3.75 ± 0.33 K/µL) in control/placebo, respectively.

White blood cell indices
The results regarding the white blood cell indices (Table 4) showed that diets imparted non-significant effect on white blood cell count and values were in the range of 10.10 ± 0.20 to 10.38 ± 0.36 K/μL. The mean values for lymphocytes (%) ranged from 78.53 ± 4.88 to 82.00 ± 7.30%. The maximum neutrophiles percentage was 14.13 ± 0.40% in D 1 (placebo) while the least neutrophiles percentage 13.23 ± 0.80% was in D 3 (N. sativa essential oil). The non-significant variations among the mean values for diets regarding monocytes percentage ranged from 2.74±0.06 to 2.82±0.10%. Eosinophiles and basophiles varied non-significantly from 1.26 ± 0.11 to 1.75 ± 0.15% and 0.64± 0.10 to 0.77 ± 0.10, respectively.

Discussion
Researchers across the globe are more interested in probing the safety assessments and toxicological considerations of traditional Means sharing same letters in a column/row do not differ significantly at P < 0.05 medicines. A well-defined research can provide evidences about dietary/health claims associated with some traditional foods or medicines. The addition of N. sativa fixed and essential oil increased feed and water intakes as compared to control group. Groups of rats fed on experimental diets gained higher weights as compared to control. The declining tendency in the body weight during oxidative stress conditions is in well agreement with the findings of (Kanter et al., 2009). These undesirable effects are due to potassium bromate. N. sativa fixed and essential oils modulated organ to body weight ratio significantly. According to    (Ramadan, 2007), antioxidant fractions of N. sativa can be employed to improve the antioxidant status of the body and this might be the possible reason for normal ranges of organ to body weight ratio in groups fed on diets containing N. sativa fixed and essential oils.
Hypercholesterolemia is an allied consequence of oxidative stress and characterized by elevated levels of cholesterol and triglycerides. In the present research, we observed higher values for cholesterol and triglycerides as compared to normal. In contrary, experimental diets were effective in improving lipid profile as evident from 14.24 and 8.27% decrease in cholesterol in N. sativa fixed and essential oils groups, respectively. The experimental diets showed lipid lowering potential and improved antioxidant status. In the present study, blood glucose increased by 18.86% in control, while same trait decreased by 7.75 and 9.47% in NSFO and NSEO groups, respectively. Insulin secretion tends to drop in stress condition by 13.01, while improved by 6.32 and 11.45% in D 2 and D 3 groups, respectively. The findings highlighted the prospects of using appropriate antioxidant rich food to overcome the adverse consequences of oxidative stress including hypercholesterolemia and hyperglycemia (Khalaji et al., 2011).
In the previous study, we observed higher AST, ALT, and ALP activities in oxidative stress (Sultan et al., 2012). The modulation of hematological and serological attributes in oxidative stressed conditions is due to antioxidant perspectives and bioactive components. Such outburst in the activities could further lead to damage to endothelial membranes. Thymoquinone, carvacrol, t-anethol and 4-terpineol are important antioxidants found in N. sativa mainly responsible in mitigating oxidative stress. The studies conducted to counteract the CCl 4 damage provided evidence about the protective action of N. sativa or its active ingredient thymoquinone, owing to its ability to inhibit lipid peroxidation (Yaman et al., 2010). Oxidative stress disturbs the hematological indices, decreasing the red & white blood cells along with decreasing hemoglobin levels. However, the bioactive molecules present in N. sativa holds the potential as antioxidant and thus could be useful for treating ailments characterized with oxidative stress.

Conclusion
Concluding, N. sativa fixed and essential oils exhibited strong antioxidant activity, mitigated the oxidative stress, and improved various serological and hematological attributes. The oxidative stress influenced the hematological attributes negatively but experimental diets were slightly successful to normalize the values. Comprehensive studies (cohort and community based trials) are required to test their effectiveness as nutraceuticals in human subjects.