Influence of Black Cumin Fixed and Essential Oil Supplementation on Markers of Myocardial Necrosis in Normal and Diabetic Rats
Muhammad Tauseef Sultan,
Masood Sadiq Butt,
Faqir Muhammad Anjum
The cardiovascular disorders are major cause of mortality in diabetic patients and myocardial necrosis, atherosclerosis and high blood pressure are major ailments in this regard. The core objective of the present research study was to explore the role of Black Cumin Seed Fixed Oil (BCFO) and Black Cumin Essential Oil (BCEO) on the markers of myocardial necrosis i.e. cardiac enzymes in normal and diabetic Sprague dawley rats. Diabetes mellitus was induced using streptozotocin @ 60 mg/Kg body weight. The three diets were prepared i.e. D1 (control), D2 (BCFO @ 4.0%) and D3 (BCEO @ 0.30%) and added separately in the diets and fed to the respective groups for a period of 56 days. The results indicated that diets were insignificant and level of cardiac enzymes remained in the safe ranges in normal rats. However, diabetes affected deleteriously and markers of myocardial necrosis increased in control group. In comparison, BCFO and BCEO normalized the levels of markers of myocardial necrosis. However, BCEO was more effective in reduced the elevated levels of these enzymes. Moreover, increased levels of transferases in diabetic rats were also reduced as a function of BCFO and BCEO treatment. In the nutshell, it can be assumed that black cumin essential oil is more effective in reducing the elevated levels of enzymes thus reducing the onset of cardiovascular disorders in diabetes mellitus.
Cardiovascular Disorders (CVD) are major cause of mortality and morbidity over the globe. The reasons behind are lack of physical activity, inadequate diets and hypertension (Butt et al., 2009) Sometime, diseases like diabetes mellitus are also accompanied with CVD incidence and according to estimates 50% of diabetic patients are died due to ill heart health. Diabetes mellitus induced metabolic changes leading to myocardial necrosis, cardiac hypertrophy owing to accumulation of cholesterol, triglycerides, phospholipids and glycated protein in the myocardium (Christopher et al., 2003).
Induction of diabetes mellitus increases levels of cholesterol, low density lipoprotein and total triglycerides while reducing high density lipoprotein contents (Kanter et al., 2003; Meral et al., 2001; Altan et al., 2007; El-Nekeetya et al., 2007). It is being hypothesized that the cholesterol deposition due to increased activities of cholesterol synthesizing enzyme and reduced activities of antioxidants leads to Low Density Lipoproteins (LDL) oxidation (Singh et al., 2007). Moreover, the depletion of enzymes due to increased free radical concentration could be considered one of the important complications of diabetes mellitus (Clarkson, 1995). Numerous studies have reported that the antioxidant supplementation could avert the chances of such malfunctioning of key humans systems. Tocopherol serve as an effective agent for reducing the extent of damage caused by free radicals along with its cholesterol lowering potential (Eder et al., 2002). Moreover, Van Acker et al. (2000) highlighted the importance of antioxidant and enumerated that they are equally effective as tocopherols in lowering the diabetes complications (Sesso et al., 2003).
Plants are naturally bestowed with variety of components commonly known as phytochemicals that holds potential to cure various maladies (Butt et al., 2008). Black cumin is one such example with ample of phytochemicals mainly concentrated in its fixed and essential oils (Sultan et al., 2009). The tocopherols and sterols in fixed oils and antioxidant and alkaloids in essential oils can be effective utilized in lowering the elevated levels of markers of myocardial necrosis. The improvement in serum lipid profile can also be beneficial in reducing diabetes complication like cardiovascular disorders resulting due to increased susceptibility towards atherosclerosis and atherogenesis.
MATERIALS AND METHODS
Black cumin seeds of indigenous variety were obtained from Barani Agricultural Research Institute (BARI), Chakwal. Raw materials were procured from local market while reagents (analytical and HPLC grade) and standards were purchased from Sigma-Aldrich (Sigma-Aldrich Tokyo, Japan) and Merck (Merck KGaA, Darmstadt, Germany). Sprague dawley rats were procured from National Institute of Health (NIH), Islamabad, Pakistan.
Extraction of fixed and essential oil: The oil from the black cumin seed was extracted through solvent extraction technique as described in AOCS (1998); hexane used as solvent and after extraction was recovered by Rotary Evaporator (Eyela, Japan). Essential oil was extracted following the method of Kanter et al. (2003) using locally fabricated hydro-distillation apparatus. The extracted black cumin fixed oil was administered @ 4.0% and black cumin essential oil @ 0.30% to check their potential to reduce myocardial necrosis. The doses were selected on the basis of preliminary trials. The both oils were added in the diets and further fed to their respective groups for a period of 56 days.
Test animals and their housing: Sprague dawley rats were used during the present investigation as test animals. A total of 60 rats 6-7 weeks old, were selected after physical and behavioural examination. The animals were acclimatized by feeding basal diet for a period of one week. They were divided into 2 groups of 30 further divide into three of 10 rats each. The diets prepared from the selected treatments were fed for a period of 8 weeks. The experimental diets contained fixed amounts of oil (10%), protein (20%), corn starch (55%) and cellulose (10%). The mineral and vitamin mixtures (AIN-76) were added @ 3% and 1%, respectively.
Diabetic mellitus was induced in rats (Weight 150-200 grams) by injecting Streptozotocin (STZ) @ 60 mg/Kg body weight, dissolved in 0.01 M citrate buffer (pH 4.5), intravenously. The blood glucose of each rat was monitored after injecting STZ to check the glucose response.
The animals were maintained according to standard guidelines of Animal Institute of Nutrition (AIN), USA. Feed and water intakes were monitored on daily basis, while body weights were recorded weekly throughout the experimental period. Half of the overnight fasted rats were sacrificed after four weeks of feeding trail and the rest at the end of study. Blood samples were collected through cardiac puncture and serum was collection following the method of Uchida et al. (2001).
Biomarkers of myocardial necrosis: The activity of LDH was determined using a commercially available kit from Biocon® (Germany). Likewise, Creatine Phosphokinase (CPK) were estimated using commercial kits from Cayman Chemicals (Kairisto et al., 1993) and serum creatine phosphokinase-MB (CK-MB) by using commercial kit from Stanbio Laboratory (Boerne, TX).
Biomarkers of multiple organ toxicity: Activities of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) and Alkaline Phosphatase (ALP) were determined by the enzymatic methods using commercial kits and used as biochemical markers for the early acute damage (ChronoLab, Switzerland). Total bilirubin contents were also assayed using commercial kits.
Statistical analysis: Data obtained was analyzed statistically using statistical package i.e. Cohort V-6.1 (Co-Stat Statistical Software, 2003). Sample for each analysis was run quadruplet and values expressed are means ± standard deviation. The distribution of the data was checked and analysis of variance was applied afterwards. The significant differences were further compared through Duncans Multiple Range test (DMRt).
Statistical analysis demonstrated that cardiac enzymes Lactate Dehydrogenase (LDH), creatine kinase and its isoenzyme MB (CPK and CK-MB) were affected non-significantly in relation to diets, study intervals and their interactions (Table 1). The means for LDH indicated that values were in the range of 340.89±17.15 to 382.38±15.15mg/dL. There was less activity of this enzyme in the group of rats fed on black cumin fixed oil as compared to control and black cumin essential oil groups. The similar patterns of results were recorded for the creatine kinase and its isoenzyme MB (CPK and CK-MB). Both these enzymes varied from 161.44±8.50 to 191.44±12.29 mg/dL and 20.53±1.08 to 29.41±1.32 mg/dL, respectively. Overall, the values for cardiac enzymes remained in the normal ranges as described in the literature.
Effect of diets and study intervals on cardiac enzymes in normal
|NS = Non-significant,LDH = Lactate dehydrogenase
CPK = Creatine phosphokinaseCK-MB = Creatine kinase-MB
It is observed from Table 2 that diets, study intervals and
their interactions exhibited non-significant differences in liver enzymes (alanine
aminotransferase, ALT; aspartate aminotransferase, AST; alkaline phosphatase,
ALP) and bilirubin. Activities of liver enzymes like AST, ALT and ALP ranged
from 116.00±2.62 to 120.67±4.03, 43.06±0.76 to 45.88±1.28
and 169.65±4.54 to 186.84±6.30 IU/L, respectively in different
diets groups. Moreover, bilirubin proteins varied non-significantly from 0.64±0.03
to 0.66±0.05 mg/dL.
Induction of diabetes by means of Streptozotocin injection resulted in marked
increase in the activities of the biomarkers of myocardial necrosis. The diabetes
mellitus is well characterized with the high levels of glucose and low level
of insulin. Moreover, such abnormalities are more prominent when we look into
of diets and study intervals on liver and kidney function tests in normal
NS = Non-significantAST = Aspartate aminotransferaseALT = Alanine aminotransferaseALP
= Alkaline phosphatase
of diets and study intervals on cardiac enzymes in diabetic rats
|NS = Non-significant
The death count in diabetic patients is often due to CVD. In the present investigation,
data pertaining to cardiac enzymes when subjected to statistical analysis (Analysis
of variance) revealed that cardiac enzymes were significantly affected only
as a function of study duration while diets and interaction did not imparted
significant impact on same enzymes (Table 3). Cardiac enzymes
LDH, CPK and CK-MB remained in the ranges of 365.36±16.22 to 610.35±30.94
mg/dL, 189.67±8.42 to 331.21±9.63 mg/dL and 28.17±1.25
to 36.53±1.85 mg/dL, respectively (Table 4). Cardiac
enzymes concentrations increased with the passage of time and all enzymes gave
maximum concentration at the end of study (56 days) while minimum values were
recorded at the start of the study (Table 4) with the exception
of CK-MB. It was further observed that groups of rats fed on control diets were
shown increasing tendency for levels of cardiac enzymes, while black cumin fixed
and essential oils attenuated the adverse situation and elevated levels of these
enzymes due to induction of diabetes mellitus were normalized. These results
indicated the possibility of utilization of these phytochemicals rich fraction
in reducing the extent of myocardial necrosis.
It was deduced from mean squares (Table 5) that diets, study duration and their interaction affected liver enzymes significantly. Means (Table 6) indicated that control groups showed maximum values i.e. 131.35±2.685, 65.70±2.529, 326.83±62.192 IU/L and 1.09±0.089 mg/dL for AST, ALT, ALP and total bilirubin contents, respectively. Diets containing black cumin fixed and essential oils mitigated the adverse consequences of diabetes mellitus significantly; AST reduced from 126.05±5.273 to 102.88±4.531 and 128.03±7.388 to 97.66±4.951 IU/L whereas ALT from 65.87±2.756 to 54.56±2.403 and 70.98±4.096 to 46.81±2.373 IU/L, respectively. Moreover, ALP activity i.e. 251.36±22.654 and 238.46±21.717 IU/L was recorded for black cumin fixed and essential oils group, respectively. Whereas total bilirubin contents in these groups were found to be 0.86±0.041 and 0.82±0.026 mg/dL, correspondingly.
Nutrients present in the diets e.g. macro and micronutrients plays important role in normal functionality of the organisms (Mahmood et al., 2008). The plants are important sources of several key nutrients. In the recent era consensus has been sought on the development of functional and nutraceuticals foods from plants origin for the treatments of various disorders. Prevention of diabetes mellitus and its complications is important target of such modern innovations. In this regard, animal modeling for safety evaluation, toxicity limits and healthiness of functional and nutraceutical foods are imperative before dissemination of knowledge to end users. Malley et al. (2007) suggested clinical pathological evaluation by means of blood biochemical screening as one of the safety assessment tools when some novel food sources are exploited for their appraisal as safe human food ingredient (Singh et al., 2002). Sprague dawley rats are special strains of rats often used for safety assessment of food and rat modeling to study the effects of diets, special foods or efficacious study of bioactive compounds. In the present research investigation, same strain of rats was used in rat modeling studies to check the influence of black cumin seed fixed and essential oils in ameliorating the levels of cardiac and liver enzymes.
In the present investigation, diets containing black cumin fixed and essential oil did not change cardiac enzymes in normal rats and all values remained in the normal ranges. Morita et al. (2008) reported the normal values for the Sprague dawley rats and findings of present investigation were remained in the safe limits (Chengelis et al., 2008).
Levels of cardiac enzymes and liver enzymes are important biomarkers of myocardial necrosis and their abnormal values indicate possible toxicity symptoms (Farag et al., 2006; Patel et al., 2008). It is concluded from the findings of present research that all these parameters remained in the normal ranges as described in safety studies conducted by Chengelis et al. (2008) and Morita et al. (2008). They reported that values of ALP, ALT and AST usually lie in the range of 139-260, 33-81 and 88-162 U/L respectively (Petterino and Argentino-Storino, 2006).
Streptozotocin induced diabetes mellitus results in abnormal values for kidney
and livers enzymes. This phenomenon is attributed to free radical production
that causes membrane damage especially in the liver and kidney tissues (Daba
and Abdel-Rahman, 1998).
of cardiac enzymes in diabetic rats
|D1 = Control diet; D2 = Black cumin
seed fixed oil; D3 = Black cumin seed essential oil
of diets and study intervals on liver function tests in diabetic rats
|NS = Non-significant; * = Significant; ** = Highly significant
AST = Aspartate aminotransferaseALT = Alanine aminotransferaseALP = Alkaline
and kidney functions tests in diabetic rats
|D1 = Control diet; D2 = Black cumin
seed fixed oil; D3 = Black cumin seed essential oil.
Means sharing same letters in a column/row do not differ significantly at
In the present investigation, values for AST, ALT, ALP and total bilirubin
were observed higher in control group as compared to lower in black cumin fixed
and essential oils groups. The reduction in oxidative damage and improvement
in antioxidant status of the body are possible due to influence of the experimental
diets. Improvement in antioxidant status of the body certainly assures the normal
functioning of the human body (Kökdil et al., 2006). Black cumin
fixed oil is rich in tocopherols whereas thymoquinone, carvacrol, thymol, cymene,
t-anethole and 4-terpineol are the major antioxidants of black cumin essential
oil (Wajs et al., 2008). Antioxidant potential of experimental diets
is certainly due to these functional ingredients. These findings are in agreement
with Kanter et al. (2003), Zaoui et al. (2002) and Meral et
al. (2001). Similarly, Shinde and Goyal (2003) reported significant decrease
in creatinine, urea, AST and ALT levels in diabetic rats treated with antidiabetic
agents (Sahin et al., 2007).
Conclusion: Myocardial necrosis is one of key cardiovascular disorder and its prevalence in diabetes mellitus is often attributed to LDL oxidation, free radical production and oxidative stress that cause myocardial membrane damage. Conclusive approach drawn from this part of efficacy study is black cumin fixed and essential oils hold potential to attenuate the elevated levels of biomarkers of myocardial necrosis. It was further observed that diets improved the health of normal rats while brought balance in the level of cardiac and liver enzymes. The findings should be tested in Cohort studies in human diabetic subjects for meticulousness.
Altan, M.F., M. Kanter, S. Donmez, M.E. Kartal and S. Buyukbas, 2007.
Combination therapy of Nigella sativa
and human parathyroid hormone on bone mass, biomechanical behavior and structure in streptozotocin-induced diabetic rats. Acta. Histochemica., 109: 304-314.PubMed |
Official Methods and Recommended Practices of AOCS. 5th Edn., American Oil Chemical Society, Champaign, IL., USA
Butt, M.S., A. Nazir, M.T. Sultan and K. Schroen, 2008. Morus alba
L.: Natures functional tonic. Trends Food Sci. Technol., 19: 505-512.Direct Link |
Butt, M.S., M.T. Sultan, M.S. Butt and J. Iqbal, 2009.
Garlic: Natures protection against physiological threats. Crit. Rev. Food Sci. Nutr., 49: 538-551.PubMed |
Chengelis, C.P., J.B. Kirkpatrick, K.S. Regan, A.E. Radovsky and M.J. Beck et al
28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats. Food Chem. Toxicol., 46: 978-989.Direct Link |
Christopher, C.L., L.N. Mathuram, G. Genitta, I. Cyrus and S.J. Sundar, 2003.
Omega-3 polyunsaturated fatty acids inhibit the accumulation of PAS-positive material in the myocardium of STZ-diabetic Wistar rats. Int. J. Cardiol., 88: 183-190.Direct Link |
Clarkson, P.M., 1995.
Antioxidants and physical performance. Crit. Rev. Food Sci. Nutr., 35: 131-141.PubMed |
Co-Stat Statistical Software, 2003.
CoHort v. 6.2, Co-Stat Statistical Software 2003. Monterey, CA.
Daba, M.H. and M.S. Abdel-Rahman, 1998.
Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol. Lett., 95: 23-29.CrossRef |
Eder, K., D. Flader, F. Hirche and C. Bransch, 2002.
Excess dietary vitamin E lowers the activities of antioxidant enzymes in erythrocytes of rats fed salmon oil. J. Nutr., 132: 3400-3404.Direct Link |
El-Nekeetya, A.A., W. El-Kholyb, N.F. Abbasc, A. Ebaidb, H.A. Amraa and M.A. Abdel-Wahhaba, 2007.
Efficacy of royal jelly against the oxidative stress of fumonisin in rats. Toxicon, 50: 256-269.Direct Link |
Farag, R.S., E.A. Mahmoud, A.M. Basuny and R.F.M. Ali, 2006.
Influence of crude olive leaf juice on rat liver and kidney functions. Int. J. Food Sci. Technol., 41: 790-798.Direct Link |
Kanter, M., I. Meral, Z. Yener, H. Ozbek and H. Demir, 2003.
Partial regeneration/proliferation of the β-cells in the islets of langerhans by Nigella sativa
L. in streptozotocin-induced diabetic rats. Tohoku J. Exp. Med., 210: 213-219.CrossRef | PubMed | Direct Link |
Kairisto, V., A. Virtanen, E. Uusipaikka, L.M. Voipio-Pulkki, V. Nanto, O. Peltola and K. Irjala, 1993.
Method for determining reference changes from patients serial data: Example of cardiac enzymes. Clin. Chem., 39: 2298-2304.Direct Link |
Kokdil, G., L. Tamer, B. Ercan, M. Celik and U. Atik, 2006.
Effects of Nigella orientalis
fixed oils on blood biochemistry in rats. Phytother. Res., 20: 71-75.PubMed |
Mahmood, S., M.S. Butt, F.M. Anjum and H. Nawaz, 2008.
Baking and storage stability of retinyl acetate (Vitamin A) fortified cookies. Pak. J. Nutr., 7: 586-589.CrossRef | Direct Link |
Malley, L., N.E. Everds, J. Reynolds, P.C. Mann and I. Lamb et al
Subchronic feeding study of DAS-59122-7 maize grain in sprague-dawley rats. Food Chem. Toxicol., 45: 1277-1292.PubMed |
Morita, O., Y. Tamaki, J.B. Kirkpatrick and C.P. Chengelis, 2008.
Safety assessment of heated diacylglycerol oil: Subchronic toxicity study in rats. Food Chem. Toxicol., 46: 2748-2757.Direct Link |
Patel, C., P. Dadhaniya, L. Hingorani and M.G. Soni, 2008.
Safety assessment of pomegranate fruit extract: Acute and subchronic toxicity studies. Food Chem. Toxicol., 46: 2728-2735.PubMed |
Petterino, C. and A. Argentino-Storino, 2006.
Clinical chemistry and haematology historical data in control Sprague-Dawley rats from pre-clinical toxicity studies. Exp. Toxicol. Pathol., 57: 213-219.Direct Link |
Sahin, K., M. Ondercib, M. Tuzcuc, B. Ustundagd and G. Cikime et al
Effect of chromium on carbohydrate and lipid metabolism in a rat model of type 2 diabetes mellitus: The fat-fed, streptozotocin-treated rat. Metab. Clin. Exp., 56: 1233-1240.CrossRef | Direct Link |
Sesso, H.D., J.M. Gaziano, S. Liu and J.E. Buring, 2003.
Flavonoid intake and the risk of cardiovascular disease in women. Am. J. Clin. Nutr., 77: 1400-1408.Direct Link |
Shinde, U.A. and R.K. Goyal, 2003.
Effect of chromium picolinate on histopathological alterations in STZ and neonatal STZ diabetic rats. J. Cell. Mol. Med., 7: 322-329.CrossRef |
Singh, A.S., D.T. Pal, B.C. Mandal, P. Singh and N.N. Pathak, 2002.
Studies on changes in some of blood constituents of adult cross-bred cattle fed different levels of extracted rice bran. Pak. J. Nutr., 1: 95-98.CrossRef | Direct Link |
Singh, B.B., S.P. Vinjamury, C. Der-Martirosian, E. Kubik and L.C. Mishra et al
Ayurvedic and collateral herbal treatments for hyperlipidemia: A systematic review of randomized controlled trials and quasi-experimental designs. Altern. Ther. Health Med., 13: 22-28.PubMed | Direct Link |
Sultan, M.T., M.S. Butt, F.M. Anjum, A. Jamil, S. Akhtar and M. Nasir, 2009.
Nutritional profile of indigenous cultivar of black cumin seeds and antioxidant potential of its fixed and essential oil. Pak. J. Bot., 41: 1321-1330.Direct Link |
Uchida, K., T. Satoh, Y. Ogura, N. Yamaga and K. Yamada, 2001.
Effect of partial ileal bypass on cholesterol and bile acid metabolism in rats. Yanago Acta Med., 44: 69-77.Direct Link |
Van Acker, F.A., O. Schouten, G.R. Haenen, W.J. Van der Vijgh and A. Bast, 2000.
Flavonoids can replace alpha-tocopherol as an antioxidant. FEBS Lett., 473: 145-148.PubMed |
Wajs, A., R. Bonikowski and D. Kalemba, 2008.
Composition of essential oil from seeds of Nigella sativa
L. cultivated in Poland. Flavour Fragr. J., 23: 126-132.CrossRef | Direct Link |
Zaoui, A., Y. Cherrah, K. Aloui, N. Mahassine, H. Amarouch and M. Hassar, 2002.
Effect of Nigella sativa
fixed oil on blood homeostasis in rat. J. Ethnopharmacol., 79: 23-26.CrossRef | Direct Link |