August 30, 2019 Archives • 2019 • vol.2 • 139-151 ANTIOXIDANT EFFECT OF HUMAN URINE IN ALLOXAN-INDUCED DIABETIC RATS 1Ogugua, Victor N.; 1,2*Okeke, Emmanuel S.; 1Anaduaka, Emeka G.; 1Ugwu, Obumneke J.; 1,2Nwankwo, Nicodemus E.; 1Okenwa, Ezinne J.; 1Ihim, Chiamaka A. and 1Enwerem, Uchechukwu 1Department of Biochemistry, University of Nigeria, Nsukka. 2Natural Science Unit, School of General Studies, University of Nigeria, Nsukka. [email protected] Abstract This work evaluated the effects of different doses of the human urine on normal and alloxan- induced diabetic rats. The experimental design comprises of five (5) groups of four (4) animals each. At the end of the treatment period, blood glucose levels, protein, MDA, catalase, Glutathione and vitamin C concentrations were determined in both the normal and diabetic rats. Oral administration of low and higher doses (5ml/kg and 10 ml/kg) of human urine for 7 days resulted in significant (p < 0.05) decrease in blood glucose and protein concentrations of the diabetic rats compared to the untreated diabetic rats. However, the catalase concentration of the rats treated with (5ml/kg and 10 ml/kg) of human urine showed a significant (p < 0.05) increase compared to the untreated diabetic rats. This results show that human urine has great potentials of increasing the ascorbic acid concentration of diabetic rats treated with standard drug, 5ml/kg and 10ml/kg of urine when compared to diabetic untreated group and with a concomitant antioxidant effect on alloxan-induced lipid peroxidation in rats. However, it was observed that there is a significant (p < 0.05) increase in the mean concentration of catalase of the groups treated with standard drug, 5ml/kg and 10ml/kg of urine when compared to diabetic untreated group. Furthermore, there was an observed significant (p < 0.05) increase in the mean concentration of glutathione and SOD of the groups treated with standard drug, 5ml/kg and 10ml/kg of urine when compared to diabetic untreated group which proves human urine to have an antioxidant property. Keywords: Diabetes; Urine; Alloxan; Glutathione; Lipid peroxidation; catalase; SOD. http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Ogugua, et al. 140 (pag 139-151) Introduction Chemicals and reagents: all chemicals and reagents were obtained from Sigma- Aldrich Diabetes mellitus is a metabolic disorder Chemical Co. (St Louis, MO, USA). All the chemicals characterized by hyperglycemia and insufficiency of used including the solvents, were of analytical secretion or action of endogenous insulin [1]. It is a grade. metabolic disorders with micro-and macrovascular complications that results in significant morbidity Treatment Material and mortality. It is considered as one of the five About 20 ml of early morning urine of a healthy leading causes of death in the world [2, 3]. In individual free from drug of any type was freshly modern medicine no satisfactory effective therapy is collected and the pH 6.2 was determined daily, and still available to cure diabetes mellitus [4]. Although administered to the experimental animals based on the etiology of this disease is not well defined, viral their body weight. infection, autoimmune disease and environmental factors have been implicated. Studies demonstrated Animals that oxidative stress and free radical are the lead or Adult Wistar rats of between 12 to 14 weeks with supporting actor in pathogenesis of diabetic average weight of 180 ± 13 g were obtained from the complications.[5, 6]. Diabetes is usually Department of Zoology and housed in the animal accompanied by increased production of free House of the Department of Biological Sciences, radicals or impaired antioxidant defenses.[5-7]. both in University of Nigeria, Nsukka. The animals There is increasing demand by patients to use were acclimatized for 7 days under standard natural products with antidiabetic activity due to environmental conditions, with a 12 hour light/dark side effects associated with the use of insulin and cycle maintained on a regular feed (Top feed; oral hypoglycemic agents [8-10]. From these facts, it grower mash) and water ad libitum. is needed to find nutrients and food with effective antioxidant activity against oxidative damage to Methods prevent various diseases. There are therefore many Experimental Protocol natural integrative therapies available to reduce A total of twenty (20) Wistar rats were divided casualties from communicative and non- into 5 groups of four (4) animals each. The animals communicative diseases [11]. A perfect example of were grouped as follows; such substance is human urine[12-14]. Along the history, people have been toying with their own Group 1: Normal rats (Normal control) urine to find the solution for various diseases. Group 2: Diabetic rats untreated (Positive control) Martha [12] stated that there is an extraordinary Group 3: Diabetic rats treated with 2.5 mg/kg body natural healing substance produced by our own weight of standard drug bodies that modern medicine science has proven to Group 4: Diabetic rats treated with 5 ml/kg body be one of the most powerful natural medicines weight of human urine known to man. Human urine contains among other Group 5: Diabetic rats treated with 10 ml/kg body substances a hormone called weight of human urine dehydroepiandrosterone (DHEA). Dehydroepiandrosterone (DHEA), a natural Induction of experimental diabetes hormone in the body, helped rebalance the set of Diabetes was induced by intraperitoneal injection of chemical reactions that falls out of balance due to 1% Alloxan (150 mg/kg b/w) in overnight – fasted chronic high blood sugar in type 2 diabetes. This animals after acclimatization. Diabetes was work was carried out to scientifically ascertain the confirmed three (3) days later in the alloxan – traditional use of human urine in treatment of many treated animal showing blood glucose of 250 mg/dL ailments and diseases. or higher were reflected to be diabetic and were encompassed in our study [15]. This day was Materials and Methods considered as the 1st day of our experiment. Chemicals Further, rats were divided into five groups (n = 4 per http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Ogugua, et al. 141 (pag 139-151) group). Group 1 received normal saline (NS) and Statistical Analysis served as control; group 2 served as diabetic Data were reported as means ± SEM, where untreated group; group 3 diabetic rats treated with appropriate. Both one- and two- way analysis of 2.5 mg/kg body weight of standard drug serving as variance (ANOVA) were used to analyze the positive control; group 4 are diabetic rats treated experimental data and Duncan multiple test range with 5 ml/kg body weight of human urine; group 5 was used to compare the group means obtained are diabetic rats treated with 10ml/kg body weight after each treatment with control measurements. of human urine. Blood sample was collected Differences were considered significant when p < through ocular puncture for further biochemical 0.05. analysis. The glucose level was determined on the Results first day, the third day and on the seventh day. After treatment, the animals were sacrificed by Figure 1. below shows the mean value of glucose decapitation and the blood was collected in tubes concentration for the normal control after seven containing EDTA. The plasma was immediately days of treatment is 103mg/dl that of diabetic separated by low-speed centrifugation (for 1500 g untreated is 342.5mg/dl. The mean glucose levels for for 15 min, 4°C). groups III (Glibenclamide), IV (5ml/kg), V (10ml/kg) are 179.5ml/dl, 113.5ml/dl and 146.25ml/dl Biochemical Analysis respectively. There was a significant decrease (p < 0.05) in the glucose concentration of the groups Glucose concentration: was measured by an treated with 5ml/kg and 10ml/kg of urine when oxygen rate method employing a Beckman Coulter compared to the diabetic untreated group. The Oxygen electrode. result also shows a significant decrease (P <0.05) in glucose concentration from day 3 to day 7 in group Determination of Proteins Concentrations: III, IV, and V. There was also more decrease in mean Protein content in tissue homogenate was glucose concentration of the group treated with measured by the method of Lowry [16]. 5ml/kg than those treated with 10ml/kg. Oxidative Stress Assessment in Hepatic Tissue. Fig. 2 below shows the mean concentrations of Oxidative stress markers were determined in the protein for the normal group is 4.60mg/dL, diabetic liver homogenate using commercial kits for reduced untreated/ is 5.40mg/dL, group treated with glutathione (GSH), and malondialdehyde (MDA) glibenclamide is 5.43mg/dL, those treated with according to the manufacturer’s instructions. 5ml/kg of urine is 5.10mg/dL, while that of those treated with 10ml/kg of urine is 4.866mg/dL. There Antioxidant Activity Assessment in Hepatic was no significant difference (p > 0.05) in the mean Tissue:Antioxidant activity in hepatic tissue was concentration of protein in the group treated with assessed in the liverhomogenate using commercial 5ml/kg of urine when compared to those of the kits for the determination of catalase (CAT), and normal group. There was also no significant super oxide dismutase (SOD) activitiesaccording to difference (p > 0.05) in the mean concentration of the manufacturer’s instructions. protein in the group treated with 10ml/kg of urine when compared to those of the normal group. Determination Vitamin C Concentrations : A Figure 3 reveals the mean concentrations of MDA quantity, 1.0 g of sample was macerated with 20 ml in normal group, Diabetic untreated, group treated of 0.4% oxalic acid and filtered. About 9 ml of with Glibenclamide, group treated with 5ml/kg of indophenols reagent was added to 1 ml of the urine, and that of those treated with 10 ml/kg of filtrate.