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

Abstract

In this era, various traditional medicinal herbs were researched for their toxicological and safety aspects. The present study focused on using Nigella sativa fixed (NSFO) and essential oil (NSEO) to mitigate the adverse consequences of oxidative stress. For the purpose, oxidative stress was induced in Sprague Dawley rats using potassium bromate injection. The effect of NSFO and NSEO on indices of serum chemistry and hematology were studied. The results indicated that potassium bromate imparted drastic changes in serum chemistry and hematology e.g. it increased the blood cholesterol and blood glucose along with decreasing the insulin secretion. Oxidative stress also influenced the hematological attributes negatively but experimental diets were slightly successful in normalizing the values. The experimental diets especially essential oil that contains significant quantities of thymoquinone modulated the adverse consequences of oxidative stress with special reference to hematology and serological attributes. In the nutshell, NSFO and NSEO hold potential to mitigate the oxidative stress, and improved various serological and hematological attributes.

Keywords:
functional foods; oxidative stress; N. sativa; antioxidant; thymoquinone

1 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., 2014Bjelakovic, G., Nikolova, D., & Gluud, C. (2014). Antioxidant supplements and mortality. Current Opinion in Clinical Nutrition and Metabolic Care, 17(1), 40-44. PMid:24241129.). Resultantly, functional and nutraceutical foods have emerged as the leading trend in the present decade (Douaire & Norton, 2013Douaire, M., & Norton, I. T. (2013). Designer colloids in structured food for the future. Journal of the Science of Food and Agriculture, 93(13), 3147-3154. http://dx.doi.org/10.1002/jsfa.6246. PMid:23716173.
http://dx.doi.org/10.1002/jsfa.6246...
). 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, 2013Butt, M. S., & Sultan, M. T. (2013). Selected functional foods for potential in disease treatment and their regulatory issues. International Journal of Food Properties, 16(2), 397-415. http://dx.doi.org/10.1080/10942912.2010.551313.
http://dx.doi.org/10.1080/10942912.2010....
). 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., 2007Cheikh-Rouhou, S., Besbes, S., Hentati, B., Blecker, C., Deroanne, C., & Attia, H. (2007). Nigella sativa L.: Chemical composition and physicochemical characteristics of lipid fraction. Food Chemistry, 101(2), 673-681. http://dx.doi.org/10.1016/j.foodchem.2006.02.022.
http://dx.doi.org/10.1016/j.foodchem.200...
). 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., 2003Nickavar, B., Mojab, F., Javidnia, K., & Amoli, M. A. R. (2003). Chemical composition of the fixed and volatile oils of Nigella sativa L. from Iran. Zeitschrift für Naturforschung C, 58(9-10), 629-631. http://dx.doi.org/10.1515/znc-2003-9-1004. PMid:14577620.
http://dx.doi.org/10.1515/znc-2003-9-100...
).

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., 2014Sultan, M. T., Buttxs, M. S., Qayyum, M. M. N., & Suleria, H. A. R. (2014). Immunity: plants as effective mediators. Critical Reviews in Food Science and Nutrition, 54(10), 1298-1308. http://dx.doi.org/10.1080/10408398.2011.633249. PMid:24564587.
http://dx.doi.org/10.1080/10408398.2011....
). 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., 2009Kanter, M., Akpolat, M., & Aktas, C. (2009). Protective effects of the volatile oil of Nigella sativa seeds on β-cell damage in streptozotocin-induced diabetic rats: A light and electron microscopic study. Journal of Molecular Histology, 40(5-6), 379-385. http://dx.doi.org/10.1007/s10735-009-9251-0. PMid:20049514.
http://dx.doi.org/10.1007/s10735-009-925...
). 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, 2013Falak, S., & Jamil, A. (2013). Expression profiling of bioactive genes from a medicinal plant Nigella sativa L. Applied Biochemistry and Biotechnology, 170(6), 1472-1481. http://dx.doi.org/10.1007/s12010-013-0281-4. PMid:23686472.
http://dx.doi.org/10.1007/s12010-013-028...
).

Previously, safety assessment and toxicological evaluation was conducted (Sultan et al., 2009Sultan, M. T., Butt, M. S., & Anjum, F. M. (2009). Safety assessment of black cumin fixed and essential oil in normal Sprague Dawley rats: Serological and hematological indices. Food and Chemical Toxicology, 47(11), 2768-2775. http://dx.doi.org/10.1016/j.fct.2009.08.011. PMid:19699773.
http://dx.doi.org/10.1016/j.fct.2009.08....
) 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.

2 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).

2.1 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.

2.2 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.

2.3 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., 2001Uchida, K., Satoh, T., Ogura, Y., Yamaga, N., & Yamada, K. (2001). Effect of partial ileal bypass on cholesterol and bile acid metabolism in rats. Yanago Acta Medica, 44, 69-77.).

2.4 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., 1982Annoni, G., Botasso, B., Ciaci, D., Donato, M., & Tripodi, A. (1982). Liquid triglycerides (GPO-PAP). Medi Diagnostic Italy. The Journal of Laboratory and Clinical Medicine, 9, 115.) and difference method was used to calculate low density lipoproteins (LDL) following the procedure of (Annoni et al., 1982Annoni, G., Botasso, B., Ciaci, D., Donato, M., & Tripodi, A. (1982). Liquid triglycerides (GPO-PAP). Medi Diagnostic Italy. The Journal of Laboratory and Clinical Medicine, 9, 115.; Assmann, 1979Assmann, G. (1979). HDL-cholesterol precipitant. Randox Labs. Ltd. Crumlin Co. Antrim, N. Ireland. Internist, 20(559), 64.), (Stockbridge et al., 1989Stockbridge, H., Hardy, R., & Glueck, C. (1989). Photometric determination of cholesterol (CHOD-PAP method). The Journal of Laboratory and Clinical Medicine, 114, 142-151. PMid:2754303.), (McNamara et al., 1990McNamara, J., Cohn, J., Wilson, P., & Schaefer, E. (1990). Calculated values for low-density lipoprotein cholesterol in the assessment of lipid abnormalities and coronary disease risk. Clinical Chemistry, 36(1), 36-42. http://dx.doi.org/10.1093/clinchem/36.1.36. PMid:2297935.
http://dx.doi.org/10.1093/clinchem/36.1....
).

2.5 Serum glucose and insulin levels

For the purpose, GOD-PAP method was followed to determine the glucose concentration of individual rats (Thomas & Labor, 1992Thomas, L., & Labor, U. (1992). Enzymateischer kinetischer colorimetrischer test (GOD-PAP). Biocon Diagnostik. Hecke, 8(34516), 169-174.). In comparison, ELISA immunoassay technique was employed to observe the values of insulin (Keilacker et al., 1987Keilacker, H., Besch, W., Woltanski, K.-P., Diaz-Alonso, J., Kohnert, K.-D., & Ziegler, M. (1987). measurement of insulin in human sera using a New RIA Kit. 2. Determination of free and total insulin—Correlations to insulin antibody Levels1. Experimental and Clinical Endocrinology & Diabetes, 90(6), 271-277. http://dx.doi.org/10.1055/s-0029-1210701. PMid:3330035.
http://dx.doi.org/10.1055/s-0029-1210701...
).

2.6 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.

2.7 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.

2.8 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., 1997Steel, R. G., Torrie, J. H., & Dickey, D. A. (1997). Principles and procedures of statistics: A biological approach. New York: McGraw-Hill.).

3 Results

The onset of 21st 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., 2012Sultan, M. T., Butt, M. S., Ahmad, R. S., Pasha, I., Ahmad, A. N., & Qayyum, M. M. N. (2012). Supplementation of Nigella sativa fixed and essential oil mediates potassium bromate induced oxidative stress and multiple organ toxicity. Pakistan Journal of Pharmaceutical Sciences, 25(1), 175-181. PMid:22186327.). The results pertaining to the different parameters are discussed herein.

3.1 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.

Figure 1
Feed & water intake and body weight of oxidative stressed rats.

3.2 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.

Table 1
Effects of NSFO and NSEO on indices pertaining to hypercholesterolemia and hyperglycemia organs to body weight ratio in oxidative stressed Sprague dawley rats.

3.3 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.

Table 2
Effects of NSFO and NSEO on serum protein and serum electrolytes profile in oxidative stressed Sprague dawley rats Serum proteins profile.

3.4 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.

Table 3
Effects of NSFO and NSEO on red blood cell indices in oxidative stressed Sprague dawley rats in oxidative stressed rats.

3.5 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 D1 (placebo) while the least neutrophiles percentage 13.23 ± 0.80% was in D3 (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.

Table 4
Effects of NSFO and NSEO on white blood cells in oxidative stressed Sprague dawley rats induces in oxidative stressed rats.

4 Discussion

Researchers across the globe are more interested in probing the safety assessments and toxicological considerations of traditional 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., 2009Kanter, M., Akpolat, M., & Aktas, C. (2009). Protective effects of the volatile oil of Nigella sativa seeds on β-cell damage in streptozotocin-induced diabetic rats: A light and electron microscopic study. Journal of Molecular Histology, 40(5-6), 379-385. http://dx.doi.org/10.1007/s10735-009-9251-0. PMid:20049514.
http://dx.doi.org/10.1007/s10735-009-925...
). These undesirable effects are due to potassium bromate. N. sativa fixed and essential oils modulated organ to body weight ratio significantly. According to (Ramadan, 2007Ramadan, M. F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.): an overview. International Journal of Food Science & Technology, 42(10), 1208-1218. http://dx.doi.org/10.1111/j.1365-2621.2006.01417.x.
http://dx.doi.org/10.1111/j.1365-2621.20...
), 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 D2 and D3 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., 2011Khalaji, S., Zaghari, M., Hatami, K., Hedari-Dastjerdi, S., Lotfi, L., & Nazarian, H. (2011). Black cumin seeds, Artemisia leaves (Artemisia sieberi), and Camellia L. plant extract as phytogenic products in broiler diets and their effects on performance, blood constituents, immunity, and cecal microbial population. Poultry Science, 90(11), 2500-2510. http://dx.doi.org/10.3382/ps.2011-01393. PMid:22010235.
http://dx.doi.org/10.3382/ps.2011-01393...
).

In the previous study, we observed higher AST, ALT, and ALP activities in oxidative stress (Sultan et al., 2012Sultan, M. T., Butt, M. S., Ahmad, R. S., Pasha, I., Ahmad, A. N., & Qayyum, M. M. N. (2012). Supplementation of Nigella sativa fixed and essential oil mediates potassium bromate induced oxidative stress and multiple organ toxicity. Pakistan Journal of Pharmaceutical Sciences, 25(1), 175-181. PMid:22186327.). 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 CCl4 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., 2010Yaman, I., Durmus, A., Ceribasi, S., & Yaman, M. (2010). Effects of Nigella sativa and silver sulfadiazine on burn wound healing in rats. Veterinarni Medicina, 55(12), 619-624. http://dx.doi.org/10.17221/2948-VETMED.
http://dx.doi.org/10.17221/2948-VETMED...
). 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.

5 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.

  • Practical Application: N. sativa is widely used as medicinal plant throughout the world. The study in hand proves that it can be used in the treatment of various diseases and ailments. The industry can use it to prepare different herbal medicine due to its various pharmacological actions.

References

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  • Assmann, G. (1979). HDL-cholesterol precipitant. Randox Labs. Ltd. Crumlin Co. Antrim, N. Ireland. Internist, 20(559), 64.
  • Bjelakovic, G., Nikolova, D., & Gluud, C. (2014). Antioxidant supplements and mortality. Current Opinion in Clinical Nutrition and Metabolic Care, 17(1), 40-44. PMid:24241129.
  • Butt, M. S., & Sultan, M. T. (2013). Selected functional foods for potential in disease treatment and their regulatory issues. International Journal of Food Properties, 16(2), 397-415. http://dx.doi.org/10.1080/10942912.2010.551313
    » http://dx.doi.org/10.1080/10942912.2010.551313
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    » http://dx.doi.org/10.1016/j.foodchem.2006.02.022
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    » http://dx.doi.org/10.1002/jsfa.6246
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    » http://dx.doi.org/10.1007/s12010-013-0281-4
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    » http://dx.doi.org/10.1055/s-0029-1210701
  • Khalaji, S., Zaghari, M., Hatami, K., Hedari-Dastjerdi, S., Lotfi, L., & Nazarian, H. (2011). Black cumin seeds, Artemisia leaves (Artemisia sieberi), and Camellia L. plant extract as phytogenic products in broiler diets and their effects on performance, blood constituents, immunity, and cecal microbial population. Poultry Science, 90(11), 2500-2510. http://dx.doi.org/10.3382/ps.2011-01393 PMid:22010235.
    » http://dx.doi.org/10.3382/ps.2011-01393
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    » http://dx.doi.org/10.1515/znc-2003-9-1004
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Publication Dates

  • Publication in this collection
    09 Oct 2020
  • Date of issue
    Apr-Jun 2021

History

  • Received
    31 Jan 2020
  • Accepted
    02 Mar 2020
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