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Hematological and biochemical changes in diabetic rats fed with fiber-enriched cake
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Journal of Acute Medicine 3 (2013) 39e44 www.e-jacme.com
Original Research
Hematological and biochemical changes in diabetic rats fed with fiber-enriched cake Ochuko L. Erukainure a,b,c,*, Osaretin A.T. Ebuehi b, Folasade O. Adeboyejo a, Muhammad Aliyu c, Gloria N. Elemo a a
Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria b Department of Biochemistry, University of Lagos, Idi Araba, Lagos, Nigeria c Hussain Ebrahim Jamal (H.E.J.) Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Received 28 November 2012; accepted 5 March 2013 Available online 14 May 2013
Abstract Background: There have been emerging interests in functional foods, which fall in the gray area between conventional foods and medicine, over the years owing to their health benefits. In this study, the effect of fiber-enriched cake, produced from selected fruits, on the hematology and serum biochemistry of diabetic rats was investigated. Materials and methods: Diabetes was induced by an intraperitoneal injection of alloxan. Treatment lasted for 14 days, after which the rats were sacrificed humanely. Blood was collected by cardiac puncture and some of it was centrifuged to obtain serum. The serum was analyzed to evaluate alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, urea, albumin, and lipid profiles. The uncentrifuged blood was analyzed for hematological profile. Results: The rats fed on the cake had increased levels of red blood cells, hemoglobin, packed cell volume, red blood cells, and platelets compared to the untreated diabetic rats. Elevated levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, urea, and albumin were observed in the diabetic rats; this was significantly reduced in the cake-fed group. Induction of diabetes led to increased levels of cholesterol, low-density lipoprotein, and triglyceride, and a low level of high-density lipoprotein. These results were significantly reversed, except for the triglyceride level, in the cake-fed group. Conclusion: These results indicate the potentials of the fiber-enriched cake as a functional food with a therapeutic effect against hematological and biochemical changes associated with diabetes mellitus. Consequently, such a cake can serve as an adjunct to dietary therapy for diabetes. Copyright Ó 2013, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved. Keywords: Dietary fibers; Function enzymes; Functional food; Hypocholesteremia
1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by abnormalities in carbohydrate, protein, and lipid metabolism. This often occurs as a result of inadequate insulin release resulting in type 1 diabetes or insulin insensitivity as seen in type 2 diabetes.1 If left uncontrolled, DM can lead to hyperglycemia and increase the risks of atherosclerosis, * Corresponding author. Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria. E-mail address: [email protected] (O.L. Erukainure).
thereby leading to cardiovascular complications.2 Other complications include retinopathy, nephropathy, and neuropathy.3 Most pharmacological treatments of DM are based on either oral hypoglycemics and/or insulin therapy, which has many side effects such as weight gain, hypoglycemia, gastrointestinal disturbances, and hypersensitivity reactions.4 These have been of tremendous interest to health practitioners. Alternative therapies, especially those involving medicinal plants, for management of DM has long been in practice.5 These plants are rich sources of phytochemicals and dietary fibers, which serve as nutraceuticals and ingredients for functional foods.5
2211-5587/$ - see front matter Copyright Ó 2013, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jacme.2013.03.001
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O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44
There have been emerging interests in functional foods, which fall in the gray area between conventional foods and medicine, over the years owing to their health benefits.6 Functional foods are foods that are enriched with nutraceuticals derived from natural foods and can be added to other foods to impart specific health benefits.7 Of particular interest are the dietary fibers. The health benefits of dietary fibers have already been reported.8,9 Slavin10 reported that consistent consumption of 30e50 g/d of dietary fibers from whole food sources assists in lowering blood glucose concentrations. Galisteo et al11 showed that dietary fiber improves insulin sensitivity in individuals with type II diabetes. This paper reports the effects of a fiber-enriched cake on the hematology and serum biochemistry of alloxan-induced diabetic rats that fed on such a cake. 2. Materials and methods 2.1. Plant materials Fruits such as banana (Musa species), oranges (Citrus Sinensis L.), watermelon (Citrullus lanatus), pineapple (Ananas comosus), and pawpaw (Carica papaya) were identified to have high fiber contents. The fruits used for the study were purchased from the Ketu Fruit Market, Ketu, Lagos, Nigeria. They were rinsed with tap water and peeled. Juice was extracted from oranges, pineapples, and watermelons using a juice extractor, leaving behind the fibers. The amount of each fiber weighed was 400 g, which were blended together with pawpaw and banana (400 g each) in a warring blender for 10 minutes to produce a fiber paste. 2.2. Preparation of a high-fiber cake A fiber-enriched cake was produced according to the method described by Erukainure et al.12 Wheat flour (500 g) was taken into a plastic bowl, to which 400 g of the fiber paste was added. In addition, 100 g of margarine, 12 g of baking powder, and two eggs were added to this mixture. They were mixed with a mixer to form a dough. The dough was transferred into two baking pans greased with margarine and allowed to bake in an oven for 30 minutes at a temperature of 150 C. After baking, the cakes were allowed to cool and removed from the pans. They were wrapped in a foil paper and stored at 4 C.
2.4. Phytochemical analysis To have an idea of the active constituents of the formulated cake, its phytochemical properties were determined using standard methods.14e17 2.5. Animals Eighteen male albino rats of Wister strain, weighing about 150e200 g, were used for the study. They were fed on standard rat pellet diet and allowed to adapt for 1 week. They were provided with water ad libitum and maintained under standard laboratory conditions of natural photo period of a 12-hour lightedark cycle. Care of all animals was in accordance with the National Law on Animal Care and Use.18 The approval number from the Animal Institutional Ethical Committee is UL/CMUL/IEC 2011/1003. 2.6. Induction of diabetes Diabetes was induced by a single intraperitoneal injection of 180 mg/kg body weight of alloxan monohydrate in normal saline water in a volume of about 3 mL.12,19 After 72 hours of alloxan injection, the diabetic rats (glucose level >250 mg/dL) were separated and used for the study. 2.7. Experimental design The rats were divided into the following three groups, each consisting of six animals: Group 1dnormal rats; Group 2ddiabetic (untreated) rats; and Group 3ddiabetic þ highfiber cake-fed rats. The rats were monitored daily for food and water intake, and body weight. Their blood glucose levels were monitored on a weekly basis with a glucometer. Treatment lasted for 2 weeks. At the end of the feeding trials, the rats were fasted overnight and sacrificed humanely by cervical dislocation. 2.8. Collection of blood and preparation of serum Blood was collected from each rat with a 5 mL syringe and needle by cardiac puncture, and transferred into clean EDTA and plain centrifuge tube bottles as soon as it was collected, to prevent lysing. Part of the blood sample was centrifuged at 3000 rpm for 10 minutes, and the serum (supernatant) was transferred into labeled sample bottles. They were stored at 4 C to maintain enzyme activity.
2.3. Mineral analysis 2.9. Determination of hematological parameters Part of the cake was blended, from which 2 g was digested with concentrated nitric acid. The resulting solution was evaporated to dryness and dissolved in 100 mL deionized water. The solution was analyzed for mineral elements (calcium, potassium, iron, and zinc) using an atomic absorption spectrophotometer (AAnalyst 200, PerkinElmer) and a flame photometer (Jenway PFP7).13
Hematology profile, which covers hemoglobin level (HGB), packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC), platelets (PLT), lymphocytes (LYMP), and neutrophils (NEU), was determined using a Synchron CX5 autoanalyzer according to the manufacturer’s protocol.
O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44
2.10. Determination of serum biochemistry (liver and renal function enzymes, lipid profile)
Table 2 Phytochemical composition of fiber-enriched cake. Phytochemicals
Blood serum was used for the evaluation of biochemical parameters, including urea, creatinine, total bilirubin, conjugated bilirubin, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, using commercial kits from Randox Laboratories, UK, according to the manufacturer’s protocol. Serum’s total cholesterol, triglyceride, and high-density lipoprotein (HDL) were measured by the enzymatic colorimetric method using Randox kits. The concentration of lowdensity lipoprotein (LDL) cholesterol was calculated using the formula of Friedwald et al.20 Atherogenic Index (AI) and Atherogenic Index of Plasma (AIP) were calculated as described by Takasaki21 and Onat et al,22 respectively.
2.11. Statistical analysis To address the biological variability, each set of experiments was repeated at least three times for n ¼ 3 (phytochemical/mineral analysis) and n ¼ 5 (experimental rats). Differences between the groups were analyzed by one-way analysis of variance (ANOVA) with the aid of SPSS software (SPSS Inc., Chicago, IL, USA) standard version 17. The p values of <0.05 were considered statistically significant for differences in mean using the least of significance difference, and data were reported as mean standard deviation.
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Fiber-enriched cake 17.69 0.35 37.88 0.26 41.01 0.26
Saponin Flavonoids Phenol
Data are presented as mean SD mg/100 g; n ¼ 3.
3.3. Hematological indices Results of the hematological indices are depicted in Table 3. Induction of diabetes led to a decrease in the WBC level; a significant ( p < 0.05) reduction was also observed in the cake-fed rats. Altered levels of RBC, HGB, PCV, and PLT were observed in the untreated diabetic rats; feeding on the cake increased their levels significantly. A significant reduction was observed in the LYMP concentration of diabetic rats; feeding on the cake led to no significant increase. An increased level of NEU was observed in the untreated diabetic rats; a reduced level was observed in the cake-fed group, but it was not statistically significant. 3.4. Liver function enzymes Induction of diabetes elevated the levels of the studied liver function enzymes, except for albumin (as depicted in Fig. 1). Feeding on the fiber-enriched cake resulted in significant ( p < 0.05) reductions in the elevated levels. 3.5. Renal function enzymes
3. Results 3.1. Mineral composition Table 1 depicts the composition of the studied minerals. A very high concentration of iron and calcium was observed, with concentration of iron being the highest. Zinc concentration was observed to be the lowest.
3.2. Phytochemical properties Table 2 shows the composition of the studied phytochemicals. The saponin content of the cake was observed to be low compared to other phytochemicals. Phenol content was observed to be the highest, followed by flavonoid content. Table 1 Mineral composition of fiber-enriched cake. Minerals Calcium Zinc Iron Potassium
Fiber-enriched cake 262.09 24.57 336.17 88.41
0.46 0.24 0.16 0.32
Data are presented as mean SD mg/100 g; n ¼ 3.
Elevated levels of the studied renal function enzymes were observed in the untreated diabetic rats, as shown in Table 4. Feeding on the fiber-enriched cake led to significant ( p < 0.05) reductions in the elevated levels. Table 3 Hematology of the experimental groups. Parameters WBC (UL1 103) RBC (106/mL) HGB (g/dL) % PCV PLT LYMP (UL1 103) NEU (UL1 103) % LYMP % NEU
Group 1 7.30 1.80**,*** 6.50 11.70 33.12 445.4 4.64
Group 2 5.90 1.20*
Group 3 5.62 1.52*
0.40**,*** 5.40 0.68* 5.80 0.66* 9.30 1.29* 0.75**,*** 8.20 1.55* 1.47** 28.00 1.74*,*** 33.22 4.66** 8.66** 311.2 22.30* 364.0 27.22 0.55**,*** 3.40 0.74* 3.42 0.54*
1.49 0.70
1.98 0.50
1.70 0.83
62.06 4.65 29.10 3.72
59.32 4.72 31.72 5.36
63.28 13.18 28.30 11.50
Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3. HGB ¼ hemoglobin level; LYMP ¼ lymphocytes; NEU ¼ neutrophils; PLT ¼ platelets; PCV ¼ packed cell volume; RBC ¼ red blood cell; WBC ¼ white blood cell.
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O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44
Fig. 1. Liver function enzymes of the experimental groups. ALB ¼ albumin; ALP ¼ alkaline phosphatase; ALT ¼ alanine aminotransferase; AST ¼ aspartate aminotransferase; T BIL ¼ total bilirubin. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
Fig. 2 shows an inverse correlation between serum creatinine level and HGB (R2 ¼ 0.011). 3.6. Lipid profile Induction of diabetes led to increased levels of total cholesterol and LDL, as shown in Fig. 3. These were significantly ( p < 0.05) reduced by feeding the rats with the fiberenriched cake. Feeding on the cake was also observed to cause a significant ( p < 0.05) elevation in the HDL level. As depicted in Figs. 4 and 5, induction of diabetes led to increased AIP and AI, respectively, which were reduced significantly ( p < 0.05) in the cake-fed group. 4. Discussion
Fig. 2. Correlation between creatinine level and hemoglobin level in the experimental groups. HGB ¼ hemoglobin level.
used to prepare a cake with the aim of studying its effect on the hematological parameters and serum biochemistry of diabetic rats. The degree of anemia in diabetes patients can be associated with a number of factors, including glomerular filtration rate, urinary albumin excretion rate, and glycated h (HbA1c) levels.23 Anemia has been reported to be due to diminished erythropoietin production by failing kidneys and increased nonenzymatic glycosylation of RBC membrane proteins.24 In the present study, alterations in the RBC, HGB, and PCV levels of the diabetic rats suggest occurrence of anemia. The observed increase in these parameters on feeding the rats with the cake suggests its potency in the management of the ailment. This could be attributed to its phytochemical and mineral contents. The antioxidant properties of these phytochemicals, especially of phenols and flavonoids, have been reported in several studies.22 Their antisickling properties have also been reported.25 Thus, preventing oxidation of RBCs and HGB that often lead to hemolysis.26 It may also stimulate formation or secretion of erythropoietin in the stem cells of the animals, as evidenced by the increased level of RBC.27
Foods and nutrients play vital roles in the normal functioning of the body. Inclusion of dietary active compounds in human nutrition has been demonstrated to have tremendous health benefits and reduce the risk of chronic diseases such as colon cancer, obesity, diabetes, and diverticulosis.8,10 Their use has also been reported in the treatment and management of these chronic diseases.8,10 Dietary fiber is one such active compound.8e10 In this study, fibers from selected fruits were Table 4 Renal function enzymes of the experimental groups. Parameters (mg/dL)
Group 1
Group 2
Group 3
Uric acid Urea Creatinine
39.55 14.95** 2.92 0.43** 11.58 0.97***
57.00 12.40*,*** 6.02 3.78*,*** 13.86 5.12***
25.67 1.24* 3.92 1.68** 6.76 3.25*,**
Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
Fig. 3. Lipid profile of the experimental groups. Chol ¼ cholesterol; HDL ¼ high-density lipoprotein; LDL ¼ low-density lipoprotein; and TG ¼ triglyceride. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44
Fig. 4. Atherogenic Index of Plasma of the experimental groups. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
The reduced levels of WBC, PLT, and LYMP in diabetic rats indicate a suppression of the immune system.25,26 These cells identify and eliminate pathogens, either by attacking larger pathogens through contact or by phagocytosis.25 They form part of the innate immune system, which is also an important mediator in the activation of the adaptive immune system.28 The reduced immunity can contribute to the various complications associated with DM. Except for WBC, the cake was able to raise the others to an appreciable level compared to the control, suggesting a boost in the immune system. This increase can be attributed to the observed micronutrient qualities of the cake. It has been reported that the micronutrient deficiencies may have direct effects on the functioning of immune cells.29 Zinc has been shown to enhance the phagocytic activity of macrophages and neutrophils.30 Its supplementation for 1e2 months has been reported to restore immune responses, reduce the incidence of infections, and prolong survival.30 Iron deficiency has been associated with reversible abnormalities of immune function.31 Reduced polymorph neutrophil function has been shown to be reversed by iron administration.32
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The reduced level of the activities of the liver and renal function enzymes observed in the cake-fed groups signifies a protective potential of the cake against hepatotoxicity and renal toxicity. Increased serum levels of these enzymes have been reported in diabetic rats.32 This elevation has been attributed to the release of these enzymes from the cytoplasm into blood circulation after rupture of the plasma membrane and cellular damage.33 An increased level of creatinine has been associated with a low level of HGB, often leading to anaemia.34 The reduced creatinine level in the diabetic rats was observed to be inversely proportional to the HGB level, thus correlating with the antianemic potential of the cake. Diabetes has been associated with elevated levels of total cholesterol, triglycerides, and LDL, which are also risk factors of cardiovascular diseases.35 These elevated levels observed in diabetics have been linked to increased mobilization of free fatty acids from the peripheral fat depots.36 The observed reductions of these parameters in the cake-fed group indicate the antilipidemic potentials of the cake, which could be attributed to the fiber fortification in it. In several studies, fiber intake has been reported to be associated with higher levels of HDL and LDL in the blood.37 A mechanism put forth by Spiller38 is the increased generation of propionate, which has been shown to reduce cholesterol levels and inhibit cholesterol synthesis. Anderson et al39 also stated that reduced insulin response and altered lipid absorption are the likely factors in the effects of dietary fibers on hyperlipidemia. The low values of AI and AIP in the cake-fed rats are due to the observed high HDL level. High AIP is a sign of obesity and cardiovascular diseases.37 Thus, its low value further indicates the antilipemic potentials of the cake against diabetic dyslipidemia. In conclusion, these results indicate the potentials of the fiber-enriched cake as a functional food with therapeutic protective effects against hematological and biochemical changes associated with DM. Thus, it can serve as an adjunct to dietary therapy for diabetes. Conflicts of interest All contributing authors declare no conflict of interest. References
Fig. 5. Atherogenic Index of the experimental groups. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
1. Meral I, Donmez N, Baydas B, Belge F, Kanter M. Effect of Nigella sativa L. on heart rate and some haematological values of alloxan-induced diabetic rabbits. Scand J Lab Ani Sci. 2004;1:49e53. 2. Barar FSK. Essentials of Pharmacotherapeutics. 3rd ed. New Delhi: S. Chand and Company Ltd.; 2000. 3. Auslander W, Haire-Joshu D, Houston C, Rhee CW, Williams JH. A controlled evaluation of staging dietary patterns to reduce the risk of diabetes in African-American women. Diabetes Care. 2002;25:809e814. 4. Ono Y. Diet therapy for diabetes and obesity, considering osteoporosis. Clin Calcium. 2008;18:662e669. 5. Marles RJ, Farnsworth NR. Antidiabetic plants and their active constituents. Phytomedicine. 1995;2:137e189. 6. Kotilainen L, Rajalahti R, Ragasa C, Pehu E. Health enhancing foods: opportunities for strengthening the sector in developing countries.
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O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44 Agriculture and Rural Development Discussion Paper. 2006;30Available at, www.worldbank.org/rural. Accessed 29.02.12. Hill JO, Peters JC. Biomarkers and functional foods for obesity and diabetes. Brit J Nutr. 2002;88(suppl 2):S213eS218. Slavin JL. Dietary fiber and body weight. Nutrition. 2005;21:411e418. Nomura AMY, Hankin JH, Henderson BE, et al. Dietary fiber and colorectal cancer risk: the multiethnic cohort study. Cancer Causes Control. 2007;18:753e764. Slavin JL. Position of the American Dietetic Association: health implications of dietary fiber. J Am Diet Assoc. 2008;108:1716e1731. Galisteo M, Duarte J, Zarzuelo A. Effects of dietary fibers on disturbances clustered in the metabolic syndrome. J Nutr Biochem. 2008;19:71e84. Erukainure OL, Ebuehi OAT, Adeboyejo FO, et al. Anti-diabetic and hypoglycaemic properties of fibre-enriched cake in alloxan-induced diabetic rats. Med J Nutr Metab; 2012;. http://dx.doi.org/10.1007/s12349012-0116-9. AOAC. Official Methods of Analysis of the Association of Analytical Chemists. 16th ed. Washington, DC: AOAC; 1997. Harborne JB. Phytochemical Methods: A Guide to Modern Technique in Plant Analysis. New York: Chapman and Hall; 1993. Boham BA, Kocipai AC. Flavonoids and condensed tannins from the leaves of Hawaiian Vaccinum vaticulatum and V. calycinum. Pacific Sci. 1994;48:458e463. Ebrahimzadeh MA, Hosseinimehr SJ, Hamidinia A, Jafari M. Antioxidant and free radical scavenging activity of Feijoa sallowiana fruits peel and leaves. Pharmacologyonline. 2008;1:7e14. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A, Bekhradnia AR. Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey. Pharmacologyonline. 2008;2:560e567. Zimmerman M. Ethical guidelines for the investigation of experimental pain in conscious animal. Pain. 1983;16:109e110. Erukainure OL, Adeboyejo FO, Elemo GN, Ebuehi OAT. Modulation of lipid peroxidation, hypolipidemic and antioxidant activities in brain tissues of diabetic rats by fibre-enriched biscuits. J Acute Dis. 2012;1:42e46. Friedwald WT, Levy RT, Frederickson DS. Estimation of the concentration of low lipoprotein cholesterol in plasma without use of preparative ultracentrifuge. Clin Chem. 1972;18:499e502. Takasaki Y. Serum lipid levels and factors affecting atherogenic index in Japanese children. J Physiol Anthrop Appl Hum Sci. 2005;24: 511e515. Onat A, Can G, Kaya H, Hergenc G. Atherogenic index of plasma’ (log10 triglyceride/high-density lipoprotein-cholesterol) predicts high blood pressure, diabetes, and vascular events. J Clin Lipid. 2010;4:89e98.
23. Thomas MC, Maclsaac RJ, Tsalamandris C, et al. Unrecognized anemia in patients with diabetes: a cross sectional survey. Diabetes Care. 2003;26:1164e1169. 24. Arun GS, Ramesh KG. Improvement of insulin sensitivity by perindopril in spontaneously hypertensive and streptozotocin-diabetic rats. Indian J Pharmacol. 2002;34:156e164. 25. Palacios I, Lozano M, Moro C, et al. Antioxidant properties of phenolic compounds occurring in edible mushrooms. Food Chem. 2011;128:674e678. 26. Egunyomi A, Moody JO, Eletu OM. Antisickling activities of two ethnomedicinal plant recipes used for the management of sickle cell anaemia in Ibadan, Nigeria. Afr J Biotechnol. 2009;8:20e25. 27. Oyedemi SO, Adewusi EA, Aiyegoro OA, Akinpelu DA. Antidiabetic and haematological effect of aqueous extract of stem bark of Afzelia africana (Smith) on streptozotocin-induced diabetic Wistar rats. Asian Pac J Trop Biomed. 2011;1:353e358. 28. Bruce A, Johnson A, Lewis J, Raff M, Roberts K, Walters P. Molecular Biology of the Cell. 4th ed. New York and London: Garland Science; 2002. 29. Litman GW, Cannon JP, Dishaw LJ. Reconstructing immune phylogeny: new perspectives. Nat Rev Immunol. 2005;5:866e879. 30. Thurnham DI. Impact of disease on markers of micronutrient status. Proc Nutr Soc. 1997;56:421e431. 31. Ferencı´k M, Ebringer L. Modulatory effects of selenium and zinc on the immune system. Folia Microbiol (Praha). 2003;48:417e426. 32. Oppenheimer SJ. Iron and its relation to immunity and infectious disease. J Nutr. 2001;131:616Se635S. 33. Nwanjo HU. Studies on the effect of aqueous extract of Phyllanthus niruri on plasma glucose level and some hepatospecific markers in diabetic Wistar rats. Int J Lab Med. 2007;2:1e18. 34. Abovwe JA, Erukainure OL, Oke OV, Okafor OY, Ajiboye AJ. Modulatory effects of Globimetula braunii on lipid peroxidation and antioxidant status in hypercholesteremic rats. Invent Rapid Ethnopharmacol. 2010;1. 2010ep25. 35. Thomas S, Rampersad M. Anaemia in diabetes. Acta Diabetol. 2004;41:S13eS17. 36. Akah PA, Alemji JA, Salawu OA, Okoye TC, Offiah NV. Effects of Vernonia amygdalina on biochemical and hematological parameters in diabetic rats. Asian J Med Sci. 2009;1:108e113. 37. Bopama KN, Kanna J, Sushma G, Balaraman R, Rathod SP. Antidiabetic and antihyperlipidemic effects of neem seed kernel powder on alloxan diabetic rabbits. Indian J Pharmacol. 1997;29:162e167. 38. Spiller GA. CRC Handbook of Dietary Fiber in Human Nutrition. 2nd ed. Boca Raton, FL: CRC Press, Inc.; 1993. 39. Anderson AW, Baird P, Davis RH, et al. Health benefits of dietary fiber. Nutr Rev. 2009;67:188e205.
Journal of Acute Medicine 3 (2013) 39e44 www.e-jacme.com
Original Research
Hematological and biochemical changes in diabetic rats fed with fiber-enriched cake Ochuko L. Erukainure a,b,c,*, Osaretin A.T. Ebuehi b, Folasade O. Adeboyejo a, Muhammad Aliyu c, Gloria N. Elemo a a
Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria b Department of Biochemistry, University of Lagos, Idi Araba, Lagos, Nigeria c Hussain Ebrahim Jamal (H.E.J.) Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Received 28 November 2012; accepted 5 March 2013 Available online 14 May 2013
Abstract Background: There have been emerging interests in functional foods, which fall in the gray area between conventional foods and medicine, over the years owing to their health benefits. In this study, the effect of fiber-enriched cake, produced from selected fruits, on the hematology and serum biochemistry of diabetic rats was investigated. Materials and methods: Diabetes was induced by an intraperitoneal injection of alloxan. Treatment lasted for 14 days, after which the rats were sacrificed humanely. Blood was collected by cardiac puncture and some of it was centrifuged to obtain serum. The serum was analyzed to evaluate alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, urea, albumin, and lipid profiles. The uncentrifuged blood was analyzed for hematological profile. Results: The rats fed on the cake had increased levels of red blood cells, hemoglobin, packed cell volume, red blood cells, and platelets compared to the untreated diabetic rats. Elevated levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, urea, and albumin were observed in the diabetic rats; this was significantly reduced in the cake-fed group. Induction of diabetes led to increased levels of cholesterol, low-density lipoprotein, and triglyceride, and a low level of high-density lipoprotein. These results were significantly reversed, except for the triglyceride level, in the cake-fed group. Conclusion: These results indicate the potentials of the fiber-enriched cake as a functional food with a therapeutic effect against hematological and biochemical changes associated with diabetes mellitus. Consequently, such a cake can serve as an adjunct to dietary therapy for diabetes. Copyright Ó 2013, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved. Keywords: Dietary fibers; Function enzymes; Functional food; Hypocholesteremia
1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by abnormalities in carbohydrate, protein, and lipid metabolism. This often occurs as a result of inadequate insulin release resulting in type 1 diabetes or insulin insensitivity as seen in type 2 diabetes.1 If left uncontrolled, DM can lead to hyperglycemia and increase the risks of atherosclerosis, * Corresponding author. Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria. E-mail address: [email protected] (O.L. Erukainure).
thereby leading to cardiovascular complications.2 Other complications include retinopathy, nephropathy, and neuropathy.3 Most pharmacological treatments of DM are based on either oral hypoglycemics and/or insulin therapy, which has many side effects such as weight gain, hypoglycemia, gastrointestinal disturbances, and hypersensitivity reactions.4 These have been of tremendous interest to health practitioners. Alternative therapies, especially those involving medicinal plants, for management of DM has long been in practice.5 These plants are rich sources of phytochemicals and dietary fibers, which serve as nutraceuticals and ingredients for functional foods.5
2211-5587/$ - see front matter Copyright Ó 2013, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jacme.2013.03.001
40
O.L. Erukainure et al. / Journal of Acute Medicine 3 (2013) 39e44
There have been emerging interests in functional foods, which fall in the gray area between conventional foods and medicine, over the years owing to their health benefits.6 Functional foods are foods that are enriched with nutraceuticals derived from natural foods and can be added to other foods to impart specific health benefits.7 Of particular interest are the dietary fibers. The health benefits of dietary fibers have already been reported.8,9 Slavin10 reported that consistent consumption of 30e50 g/d of dietary fibers from whole food sources assists in lowering blood glucose concentrations. Galisteo et al11 showed that dietary fiber improves insulin sensitivity in individuals with type II diabetes. This paper reports the effects of a fiber-enriched cake on the hematology and serum biochemistry of alloxan-induced diabetic rats that fed on such a cake. 2. Materials and methods 2.1. Plant materials Fruits such as banana (Musa species), oranges (Citrus Sinensis L.), watermelon (Citrullus lanatus), pineapple (Ananas comosus), and pawpaw (Carica papaya) were identified to have high fiber contents. The fruits used for the study were purchased from the Ketu Fruit Market, Ketu, Lagos, Nigeria. They were rinsed with tap water and peeled. Juice was extracted from oranges, pineapples, and watermelons using a juice extractor, leaving behind the fibers. The amount of each fiber weighed was 400 g, which were blended together with pawpaw and banana (400 g each) in a warring blender for 10 minutes to produce a fiber paste. 2.2. Preparation of a high-fiber cake A fiber-enriched cake was produced according to the method described by Erukainure et al.12 Wheat flour (500 g) was taken into a plastic bowl, to which 400 g of the fiber paste was added. In addition, 100 g of margarine, 12 g of baking powder, and two eggs were added to this mixture. They were mixed with a mixer to form a dough. The dough was transferred into two baking pans greased with margarine and allowed to bake in an oven for 30 minutes at a temperature of 150 C. After baking, the cakes were allowed to cool and removed from the pans. They were wrapped in a foil paper and stored at 4 C.
2.4. Phytochemical analysis To have an idea of the active constituents of the formulated cake, its phytochemical properties were determined using standard methods.14e17 2.5. Animals Eighteen male albino rats of Wister strain, weighing about 150e200 g, were used for the study. They were fed on standard rat pellet diet and allowed to adapt for 1 week. They were provided with water ad libitum and maintained under standard laboratory conditions of natural photo period of a 12-hour lightedark cycle. Care of all animals was in accordance with the National Law on Animal Care and Use.18 The approval number from the Animal Institutional Ethical Committee is UL/CMUL/IEC 2011/1003. 2.6. Induction of diabetes Diabetes was induced by a single intraperitoneal injection of 180 mg/kg body weight of alloxan monohydrate in normal saline water in a volume of about 3 mL.12,19 After 72 hours of alloxan injection, the diabetic rats (glucose level >250 mg/dL) were separated and used for the study. 2.7. Experimental design The rats were divided into the following three groups, each consisting of six animals: Group 1dnormal rats; Group 2ddiabetic (untreated) rats; and Group 3ddiabetic þ highfiber cake-fed rats. The rats were monitored daily for food and water intake, and body weight. Their blood glucose levels were monitored on a weekly basis with a glucometer. Treatment lasted for 2 weeks. At the end of the feeding trials, the rats were fasted overnight and sacrificed humanely by cervical dislocation. 2.8. Collection of blood and preparation of serum Blood was collected from each rat with a 5 mL syringe and needle by cardiac puncture, and transferred into clean EDTA and plain centrifuge tube bottles as soon as it was collected, to prevent lysing. Part of the blood sample was centrifuged at 3000 rpm for 10 minutes, and the serum (supernatant) was transferred into labeled sample bottles. They were stored at 4 C to maintain enzyme activity.
2.3. Mineral analysis 2.9. Determination of hematological parameters Part of the cake was blended, from which 2 g was digested with concentrated nitric acid. The resulting solution was evaporated to dryness and dissolved in 100 mL deionized water. The solution was analyzed for mineral elements (calcium, potassium, iron, and zinc) using an atomic absorption spectrophotometer (AAnalyst 200, PerkinElmer) and a flame photometer (Jenway PFP7).13
Hematology profile, which covers hemoglobin level (HGB), packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC), platelets (PLT), lymphocytes (LYMP), and neutrophils (NEU), was determined using a Synchron CX5 autoanalyzer according to the manufacturer’s protocol.
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2.10. Determination of serum biochemistry (liver and renal function enzymes, lipid profile)
Table 2 Phytochemical composition of fiber-enriched cake. Phytochemicals
Blood serum was used for the evaluation of biochemical parameters, including urea, creatinine, total bilirubin, conjugated bilirubin, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, using commercial kits from Randox Laboratories, UK, according to the manufacturer’s protocol. Serum’s total cholesterol, triglyceride, and high-density lipoprotein (HDL) were measured by the enzymatic colorimetric method using Randox kits. The concentration of lowdensity lipoprotein (LDL) cholesterol was calculated using the formula of Friedwald et al.20 Atherogenic Index (AI) and Atherogenic Index of Plasma (AIP) were calculated as described by Takasaki21 and Onat et al,22 respectively.
2.11. Statistical analysis To address the biological variability, each set of experiments was repeated at least three times for n ¼ 3 (phytochemical/mineral analysis) and n ¼ 5 (experimental rats). Differences between the groups were analyzed by one-way analysis of variance (ANOVA) with the aid of SPSS software (SPSS Inc., Chicago, IL, USA) standard version 17. The p values of <0.05 were considered statistically significant for differences in mean using the least of significance difference, and data were reported as mean standard deviation.
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Fiber-enriched cake 17.69 0.35 37.88 0.26 41.01 0.26
Saponin Flavonoids Phenol
Data are presented as mean SD mg/100 g; n ¼ 3.
3.3. Hematological indices Results of the hematological indices are depicted in Table 3. Induction of diabetes led to a decrease in the WBC level; a significant ( p < 0.05) reduction was also observed in the cake-fed rats. Altered levels of RBC, HGB, PCV, and PLT were observed in the untreated diabetic rats; feeding on the cake increased their levels significantly. A significant reduction was observed in the LYMP concentration of diabetic rats; feeding on the cake led to no significant increase. An increased level of NEU was observed in the untreated diabetic rats; a reduced level was observed in the cake-fed group, but it was not statistically significant. 3.4. Liver function enzymes Induction of diabetes elevated the levels of the studied liver function enzymes, except for albumin (as depicted in Fig. 1). Feeding on the fiber-enriched cake resulted in significant ( p < 0.05) reductions in the elevated levels. 3.5. Renal function enzymes
3. Results 3.1. Mineral composition Table 1 depicts the composition of the studied minerals. A very high concentration of iron and calcium was observed, with concentration of iron being the highest. Zinc concentration was observed to be the lowest.
3.2. Phytochemical properties Table 2 shows the composition of the studied phytochemicals. The saponin content of the cake was observed to be low compared to other phytochemicals. Phenol content was observed to be the highest, followed by flavonoid content. Table 1 Mineral composition of fiber-enriched cake. Minerals Calcium Zinc Iron Potassium
Fiber-enriched cake 262.09 24.57 336.17 88.41
0.46 0.24 0.16 0.32
Data are presented as mean SD mg/100 g; n ¼ 3.
Elevated levels of the studied renal function enzymes were observed in the untreated diabetic rats, as shown in Table 4. Feeding on the fiber-enriched cake led to significant ( p < 0.05) reductions in the elevated levels. Table 3 Hematology of the experimental groups. Parameters WBC (UL1 103) RBC (106/mL) HGB (g/dL) % PCV PLT LYMP (UL1 103) NEU (UL1 103) % LYMP % NEU
Group 1 7.30 1.80**,*** 6.50 11.70 33.12 445.4 4.64
Group 2 5.90 1.20*
Group 3 5.62 1.52*
0.40**,*** 5.40 0.68* 5.80 0.66* 9.30 1.29* 0.75**,*** 8.20 1.55* 1.47** 28.00 1.74*,*** 33.22 4.66** 8.66** 311.2 22.30* 364.0 27.22 0.55**,*** 3.40 0.74* 3.42 0.54*
1.49 0.70
1.98 0.50
1.70 0.83
62.06 4.65 29.10 3.72
59.32 4.72 31.72 5.36
63.28 13.18 28.30 11.50
Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3. HGB ¼ hemoglobin level; LYMP ¼ lymphocytes; NEU ¼ neutrophils; PLT ¼ platelets; PCV ¼ packed cell volume; RBC ¼ red blood cell; WBC ¼ white blood cell.
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Fig. 1. Liver function enzymes of the experimental groups. ALB ¼ albumin; ALP ¼ alkaline phosphatase; ALT ¼ alanine aminotransferase; AST ¼ aspartate aminotransferase; T BIL ¼ total bilirubin. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
Fig. 2 shows an inverse correlation between serum creatinine level and HGB (R2 ¼ 0.011). 3.6. Lipid profile Induction of diabetes led to increased levels of total cholesterol and LDL, as shown in Fig. 3. These were significantly ( p < 0.05) reduced by feeding the rats with the fiberenriched cake. Feeding on the cake was also observed to cause a significant ( p < 0.05) elevation in the HDL level. As depicted in Figs. 4 and 5, induction of diabetes led to increased AIP and AI, respectively, which were reduced significantly ( p < 0.05) in the cake-fed group. 4. Discussion
Fig. 2. Correlation between creatinine level and hemoglobin level in the experimental groups. HGB ¼ hemoglobin level.
used to prepare a cake with the aim of studying its effect on the hematological parameters and serum biochemistry of diabetic rats. The degree of anemia in diabetes patients can be associated with a number of factors, including glomerular filtration rate, urinary albumin excretion rate, and glycated h (HbA1c) levels.23 Anemia has been reported to be due to diminished erythropoietin production by failing kidneys and increased nonenzymatic glycosylation of RBC membrane proteins.24 In the present study, alterations in the RBC, HGB, and PCV levels of the diabetic rats suggest occurrence of anemia. The observed increase in these parameters on feeding the rats with the cake suggests its potency in the management of the ailment. This could be attributed to its phytochemical and mineral contents. The antioxidant properties of these phytochemicals, especially of phenols and flavonoids, have been reported in several studies.22 Their antisickling properties have also been reported.25 Thus, preventing oxidation of RBCs and HGB that often lead to hemolysis.26 It may also stimulate formation or secretion of erythropoietin in the stem cells of the animals, as evidenced by the increased level of RBC.27
Foods and nutrients play vital roles in the normal functioning of the body. Inclusion of dietary active compounds in human nutrition has been demonstrated to have tremendous health benefits and reduce the risk of chronic diseases such as colon cancer, obesity, diabetes, and diverticulosis.8,10 Their use has also been reported in the treatment and management of these chronic diseases.8,10 Dietary fiber is one such active compound.8e10 In this study, fibers from selected fruits were Table 4 Renal function enzymes of the experimental groups. Parameters (mg/dL)
Group 1
Group 2
Group 3
Uric acid Urea Creatinine
39.55 14.95** 2.92 0.43** 11.58 0.97***
57.00 12.40*,*** 6.02 3.78*,*** 13.86 5.12***
25.67 1.24* 3.92 1.68** 6.76 3.25*,**
Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
Fig. 3. Lipid profile of the experimental groups. Chol ¼ cholesterol; HDL ¼ high-density lipoprotein; LDL ¼ low-density lipoprotein; and TG ¼ triglyceride. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
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Fig. 4. Atherogenic Index of Plasma of the experimental groups. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
The reduced levels of WBC, PLT, and LYMP in diabetic rats indicate a suppression of the immune system.25,26 These cells identify and eliminate pathogens, either by attacking larger pathogens through contact or by phagocytosis.25 They form part of the innate immune system, which is also an important mediator in the activation of the adaptive immune system.28 The reduced immunity can contribute to the various complications associated with DM. Except for WBC, the cake was able to raise the others to an appreciable level compared to the control, suggesting a boost in the immune system. This increase can be attributed to the observed micronutrient qualities of the cake. It has been reported that the micronutrient deficiencies may have direct effects on the functioning of immune cells.29 Zinc has been shown to enhance the phagocytic activity of macrophages and neutrophils.30 Its supplementation for 1e2 months has been reported to restore immune responses, reduce the incidence of infections, and prolong survival.30 Iron deficiency has been associated with reversible abnormalities of immune function.31 Reduced polymorph neutrophil function has been shown to be reversed by iron administration.32
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The reduced level of the activities of the liver and renal function enzymes observed in the cake-fed groups signifies a protective potential of the cake against hepatotoxicity and renal toxicity. Increased serum levels of these enzymes have been reported in diabetic rats.32 This elevation has been attributed to the release of these enzymes from the cytoplasm into blood circulation after rupture of the plasma membrane and cellular damage.33 An increased level of creatinine has been associated with a low level of HGB, often leading to anaemia.34 The reduced creatinine level in the diabetic rats was observed to be inversely proportional to the HGB level, thus correlating with the antianemic potential of the cake. Diabetes has been associated with elevated levels of total cholesterol, triglycerides, and LDL, which are also risk factors of cardiovascular diseases.35 These elevated levels observed in diabetics have been linked to increased mobilization of free fatty acids from the peripheral fat depots.36 The observed reductions of these parameters in the cake-fed group indicate the antilipidemic potentials of the cake, which could be attributed to the fiber fortification in it. In several studies, fiber intake has been reported to be associated with higher levels of HDL and LDL in the blood.37 A mechanism put forth by Spiller38 is the increased generation of propionate, which has been shown to reduce cholesterol levels and inhibit cholesterol synthesis. Anderson et al39 also stated that reduced insulin response and altered lipid absorption are the likely factors in the effects of dietary fibers on hyperlipidemia. The low values of AI and AIP in the cake-fed rats are due to the observed high HDL level. High AIP is a sign of obesity and cardiovascular diseases.37 Thus, its low value further indicates the antilipemic potentials of the cake against diabetic dyslipidemia. In conclusion, these results indicate the potentials of the fiber-enriched cake as a functional food with therapeutic protective effects against hematological and biochemical changes associated with DM. Thus, it can serve as an adjunct to dietary therapy for diabetes. Conflicts of interest All contributing authors declare no conflict of interest. References
Fig. 5. Atherogenic Index of the experimental groups. Data are presented as mean SD; n ¼ 6. *Statistically significant ( p < 0.05) as compared with Group 1. **Statistically significant ( p < 0.05) as compared with Group 2. ***Statistically significant ( p < 0.05) as compared with Group 3.
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