Pharmaceutica Acta ca Pharm a aA utic nalyt i ce Abdirahman et al., Pharm Anal Acta 2015, 6:9 http://dx.doi.org/10.4172/2153-2435.1000422 Analytica Acta ISSN: 2153-2435 Research Article Open Access Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana Abdirahman YA1*, Juma KK1, Makori WA1, Agyirifo DS1, Ngugi MP1, Gathumbi PK2, Ngeranwa JJN1 and Njagi ENM1 1 2 Department of Biochemistry and Biotechnology, School of Pure and Applied Sciences, Kenyatta University, Nairobi, Kenya Department of Veterinary Pathology, Microbiology and Parasitology, College of Agriculture and Veterinary Sciences, University of Nairobi, Nairobi, Kenya Abstract Zanha africana has been used traditionally to manage many diseases including diabetes, however, its antidiabetic activity and safety is not well evaluated. The aim of this study was to determine in vivo hypoglycemic activity and safety of the aqueous leaf extracts of this plant in male Swiss white albino mice. The antidiabetic activity was screened in alloxan induced diabetic mice using oral and intraperitoneal routes. The safety of the extract was studied in mice that were orally and intraperitoneally administered with 1 g/kg body weight of the extract daily for 28 days for 28 days by recording changes in body and organ weights, hematological and biochemical parameters. Mineral composition was estimated using total relection X-ray luorescence system and atomic absorption spectrometry. Phytochemical composition was assessed using standard procedures. The extract showed hypoglycemic activity at dose levels of 50, 100, 200 and 300 mg/kg body weight. Administration of 1 g/kg body weight of the extract decreased the body weight gain using both routes. Intraperitoneal administration of the same dose increased the organ to body weight percentages of liver, brain and kidney, and elevated white blood cell count, lymphocyte count, and levels of γ-glutamyl transpeptidase, total bilirubin and direct bilirubin and deceased levels of aspartate aminotransferase and creatinine. Increase in levels of mean corpuscular hemoglobin, γ-glutamyl transpeptidase, lactate dehydrogenase and creatine kinase, and decrease in levels of platelets, alanine transaminase, aspartate aminotransferase, urea, creatinine, total bilirubin and direct bilirubin was recorded in mice orally administered with 1 g/kg body weight of the extract. The extract contained tannins, phenols, lavonoids, saponins, and alkaloids. Sodium, Chlorine, Potassium, Calcium, Titanium, Vanadium, Chromium, Manganese, Iron, Copper, Zinc, Arsenic, Cadmium, Magnesium, Nickel and Lead were present in the extracts at levels below the recommended daily allowance. The observed hypoglycemic activity and slight toxicity could be associated with the phytochemicals and mineral/ trace elements present in this extract. Keywords: Zanha africana; Diabetes mellitus; Biochemical parameters; Hematological parameters; Hypoglycemic efect; Phytochemicals Introduction Diabetes mellitus is a metabolic disorder with increasing rates of incidence and mortality [1]. According to the International Diabetes Federation (IDF) the numbers of people sufering from diabetes are over 382 million worldwide and this number is expected to increase to over 592 million in less than 25 years [2]. he disease is characterized by hyperglycemia resulting from either defect in insulin secretion or insulin action or both [3]. Insulin is a hormone manufactured by the β-cells of the pancreas, which is required to uptake and utilize glucose as an energy source [4]. Lack of insulin or insulin resistance prevents eicient glucose uptake by most body cells except brain cells. his results in increased blood glucose levels, reduced cell utilization of glucose and increased utilization of fats and proteins as energy sources [5]. Acute, lifethreatening consequences of uncontrolled diabetes are hyperglycemia with ketoacidosis or the non-ketotic hyperosmolar syndrome. he long-term microvascular and macrovascular complications of the disease include; neuropathy (nerve damage), nephropathy (renal disease), vision disorders, cardiovascular vascular disorders, stroke and peripheral vascular diseases which can lead to ulcers, gangrene and amputation [3]. Treatment of type I diabetes requires administration of exogenous insulin so as the patient will have normal carbohydrate, protein and fat metabolism [5]. However weight gain and hypoglycemia are common side efects of insulin therapy [6]. For type II patients treatment options begin with diet and life style modiications but as disease progresses oten oral hypoglycemic agents or insulin or both are required [7]. Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Five classes of oral agents are approved for the treatment of diabetes. Although initial response may be good, oral hypoglycemic drugs may lose their efectiveness in a signiicant percentage of patients. he oral hypoglycemic drugs include; sulfonylurea, biguanide, α-glucosidase inhibitor, thiazolidinedione, and meglitinide. hese drugs have various side efects; sulfonylureas cause weight gain, biguanide cause weakness, fatigue, and lactic acidosis, α-glucosidase inhibitor may cause diarrhea while thiazolidinediones may increase LDL-cholesterol level [8]. here is a growing interest in herbal remedies to avoid the side efects associated with the conventional antidiabetic drugs [9]. he hypoglycemic action of a notable number of medicinal plants has been conirmed in animal models and non-insulin-dependent diabetic patients, and various hypoglycemic compounds have been identiied. Traditional antidiabetic plants might provide a useful source of new oral hypoglycemic compounds for development as pharmaceutical entities, or as simple dietary adjuncts to existing therapies [10]. Zanha africana is a plant remedy used by traditional health *Corresponding author: Abdirahman YA, Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya, Tel: +254726360323 / +252615202025; E-mail: hiraabe87@gmail.com Received September 11, 2015; Accepted September 28, 2015; Published September 30, 2015 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Copyright: © 2015 Abdirahman YA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 2 of 9 practitioners for treatment of many diseases such as diarrhea, typhoid fever, pneumonia, scabies, nose bleeding and to prevent and stop bleeding for women [11]. he antibacterial activity of both water and organic extracts of this plant has been demonstrated with the methanol extracts exhibiting the greatest antibacterial activity [12]. he plant has considerable antifungal activity against candida species [13]. Moreover the crude extracts of this plant caused substantial growth inhibition for Trypanosoma brucei [14]. Materials and Methods Study site his study was undertaken at the Department of Biochemistry and Biotechnology, School of Pure and Applied Sciences, Kenyatta University from December 2012 to August 2014. Kenyatta University is 23 km from Nairobi of hika Road. Collection of the plant materials and preparation of the aqueous extract he plant used in this study was collected from its natural habitat in Machakos County, Kenya. An acknowledged authority in taxonomy authenticated the botanical identity of the plant. he collected leaves of Zanha africana were let to dry under shed at room temperature for 1 month, and then ground when completely dry using an electric mill. Each one hundred grams of the powdered plant material was extracted in 1 liter distilled water at 60°C for 6 hour. he mixture was let to cool at room temperature and then decanted into dry clean conical lask through folded cotton gauze stufed into a funnel. he decanted extract was then iltered using ilter papers under vacuum pump. he iltrate was then freeze-dried for 72 hour. he freeze-dried powder was then weighed and stored in airtight container at -20°C until used for bioassay. Experimental animals he study used male Swiss White Albino mice (3-4 weeks old) that weighed 23-27 g with a mean weight of 25 g. hese were bred in the Animal house at the Department of Biochemistry and Biotechnology of Kenyatta University. he mice were housed at a temperature of 25°C with 12 hours/12 hours darkness photoperiod and fed on rodent pellets and water ad libitum. he experimental protocols and procedures used in this study were approved by the Ethics Committee for the Care and Use of Laboratory Animals of Kenyatta University, Kenya. Induction of hyperglycemia Hyperglycemia was induced experimentally by a single intraperitoneal administration of 186.9 mg/kg body weight of a freshly prepared 10% alloxan monohydrate (2,4,5,6 tetraoxypyrimidine; 5-6-dioxyuracil) obtained from Sigma (Steinhein, Switzerland) [15]. Forty-eight hours ater alloxan administration, blood glucose level was measured using a glucometer. Mice with blood glucose levels above 200 mg/dL were considered diabetic and used in this study. Prior to initiation of this experiment, the animals were fasted for 8-12 hours [16] but allowed free access to water until the end of this experiment. Experimental design he experimental design used in this study is shown in Table 1a and Table 1b. Blood sampling and glucose determination: Blood sampling was done by sterilizing the tail with 70% alcohol and then nipping the tail at the start of the experiment and repeated ater 1, 2, 3, 4, 6 and 24 hours. Bleeding was enhanced by gently “milking” the tail from the body towards the tip. Ater the operation, the tips of the tail were sterilized by swabbing with 70% ethanol. he blood glucose levels were determined with a glucose analyser model (Hypogaurd, Woodbridge, England). In vivo single dose toxicity test: he mice were randomly divided into four diferent groups of ive mice each. Group I and II consisted of untreated control mice intraperitoneally and orally, respectively, administered daily for 28 days with 0.1 ml physiological saline. Group III and IV consisted of normal mice intraperitoneally and orally administered daily for 28 days with the extract at 1 g/kg body weight in Group Description Treatment 1 Normal control Orally administered with 0.1 ml physiological saline Number of Mice 2 Diabetic control Orally administered with 0.1 ml physiological saline 5 3 Diabetic reference Orally administered with 0.075 mg glibenclamide (3 mg/kg body weight) in 0.1 ml physiological saline 5 4 Diabetic Orally administered with 1.25 mg extract (50 mg/kg body weight) in 0.1 ml physiological saline 5 5 Diabetic Orally administered with 2.5 mg extract (100 mg/kg body weight) in 0.1 ml physiological saline 5 6 Diabetic Orally administered with 5 mg extract (200 mg/kg body weight) in 0.1 ml physiological saline 5 7 Diabetic Orally administered with 7.5 mg extract (300 mg/kg body weight) in 0.1 ml physiological saline 5 5 The experimental mice were randomly divided into seven groups of ive animals each. Either 0.1 ml of physiological saline, glibenclamide or the plant extract solution was administered to each experimental mouse orally. Table 1a: Experimental design for oral administration in mice. Group Description Treatment 1 Normal control Intraperitoneally administered with 0.1 ml physiological saline Number of Mice 5 2 Diabetic control Intraperitoneally administered with 0.1 ml physiological saline 5 3 Diabetic reference Intraperitoneally administered with 0.025 Insulin units (1 IU/kg body weight) in 0.1ml physiological saline 5 4 Diabetic Intraperitoneally administered with 1.25 mg extract (50 mg/kg body weight) in 0.1 ml physiological saline 5 5 Diabetic Intraperitoneally administered with 2.5 mg extract (100 mg/kg body weight) in 0.1 ml physiological saline 5 6 Diabetic Intraperitoneally administered with 5 mg extract (200 mg/kg body weight) in 0.1 ml physiological saline 5 7 Diabetic Intraperitoneally administered with 7.5 mg extract (300 mg/kg body weight) in 0.1 ml physiological saline 5 The experimental mice were randomly divided into seven groups of ive animals each. Either 0.1 ml of physiological saline, insulin, or the plant extract solution was administered to each experimental mouse intraperitoneally. Table 1b: Experimental design for intraperitoneal administration. Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 3 of 9 0.1 ml physiological saline. During this period, mice were allowed free access to mice pellet and water and observed for any signs of general illness, change in behavior and mortality. At the end of 28 days, the mice were sacriiced. Determination of body and organ weight: he body weight of each mouse was assessed ater every seven days during the dosing period up to and including the 28th day and the day of sacriice (day zero, 7, 14, 21, 28). On the day of sacriice, all the animals were euthanized using chloroform as an inhalant anesthesia and blood samples were drawn from the heart of each sacriiced mouse. he blood samples were collected in plastic test tubes and divided into two portions. One portion was used for determination of hematological parameters. he other portion was allowed to stand for 3 hours to ensure complete clotting. he clotted blood samples were centrifuged at 3000 rpm for 10 min and clear serum samples were aspirated of and stored frozen at -20°C for metabolite and enzyme assays. he liver, kidney, heart, lungs, spleen, intestine, brain and testis were carefully dissected out and weighed. Determination of hematological parameters: Blood parameters and indices were determined using standard protocols [17]. Red blood cells count (RBC), white blood cells count (WBC), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), packed cell volume (PCV), mean corpuscular volume (MCV) and platelets (PLT) were determined in whole blood with EDTA anticoagulant using the Coulter Counter System (Beckman Coulter®, hermoFisher, UK). Diferential white blood cell count for Neutrophils, Lymphocytes, Eosinophils, Basophils and Monocytes were determined from giemsa stained blood ilms using a hemocytometer [17]. Air-dried thin blood ilms stained with giemsa stain were examined microscopically using magniication of 400 for diferential WBC counts. Determination of biochemical parameters: he biochemical parameters determined on the sera specimen using the Olympus 640 Chemistry AutoAnalyser were aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), lactate dehydrogenase (LDH), creatine kinase (CK), α-amylase (α-AMYL), total bilirubin (T-BIL), direct bilirubin (D-BIL), urea and creatinine. All reagents for the machine were commercially prepared to it the required volumes and concentrations. he reagents were in speciic containers referred to as reagent cartridges. he reagent cartridges were bar coded for the identiication by the machine. he machine was programmed for the selected tests for each sample. he sample sectors were then placed into the autoloader assembly. A number of events that occurred simultaneously were performed automatically under the direct control of the instrument microprocessor. All the assays were performed based on the standard operating procedures (SOPs) written and maintained in the Department of Laboratory Medicine, Kenyatta National Hospital. Qualitative analysis on phytochemical constituents: he extract was screened for the presence of ive major classes of phytochemicals using the recommended procedures. Alkaloids [18], Saponins [19], Flavonoids [20], Phenols [19], and Tanins [20]. Quantitative analysis on phytochemical constituents: he phytochemicals present were quantiied using standard procedures. Alkaloids [21] Saponins [22] Flavonoids [23] Phenols [24] and Tannins [25]. Mineral elements analysis: Mineral composition of the plant extract was analyzed using total relection X-ray luorescence system (TRXF) Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal and atomic absorption spectrometry (AAS). TRXF system was used to determine the content of Sodium (Na), Chlorine (Cl), Potassium (K), Calcium (Ca), Titanium (Ti), Vanadium (V), Manganese (Mn), Iron (Fe), Copper (Cu), Zinc (Zn), Gallium (Ga), Arsenic (As), Selenium (Se), Bromine (Br), Rubidium (Rb), Strontium (Sr), Nickel (Ni), Lead (Pb), and Uranium (U) in the lyophilized plant samples as described by [26]. Atomic absorption spectrometry (AAS) was used for the analysis of Magnesium, Chromium and Cadmium [27]. All the analysis were processed following the instructions from the manufacturer. Data management and statistical analysis: he Data was entered in the Microsot Excel Spread Sheet, cleaned and then exported to Statistical Package of Social Sciences (SPSS) Sotware for analysis. Results were expressed as Mean ± Standard Deviation (SD) of the number of animals used per every study point. Statistical analysis were done using ANOVA and post-ANOVA to compare the means of untreated normal control mice with diabetic mice treated with saline, diabetic mice treated with the conventional drug, and diabetic mice treated with plant extract at doses of 50 mg/kg body weight, 100 mg/ kg body weight, 200 mg/kg body weight, and 300 mg/kg body weight. For in vivo toxicity test student unpaired t-test was used to compare the data of normal control group with the group treated with the extract. p ≤ 0.05 was considered statistically signiicant. Results Efect of oral and intraperitoneal administration of aqueous leaf extracts of Zanha africana on blood glucose levels in alloxan induced diabetic mice he dry powder of Zanha africana yielded 3.45% (w/w) aqueous leaf extract. Oral administration of aqueous leaf extracts of Zanha africana at the four therapeutic dose levels (50, 100, 200 and 300mg/kg body weight) decreased the blood glucose levels from the 1st hour to the 6th hour in a dose independent manner. hereater, there was a gradual increase up to the 24th hour (Table 2, Figure 1). During the 1st hour, the percent reductions in the blood glucose levels by the four aqueous leaf extract doses were 37.87%, 10.38%, 14.76%, and 14.06%, respectively, compared to reference drug glibenclamide which lowered blood glucose levels by 8.87% within the same hour. In this hour, the four tested dose levels did not lower blood glucose levels to normal. In the 6th hour, the percent blood glucose reductions by the four aqueous leaf extract doses were 64.89%, 29.77%, 59.78% and 58.19% respectively, compared to glibenclamide which lowered blood glucose levels by 77.13% within the same hour. In this hour, the dose level 50 mg/kg body weight decreased blood glucose levels to normal and was efective as glibenclamide. However the dose levels 100 mg/kg body weight, 200 mg/kg body weight and 300 mg/kg body weight did not lower blood glucose levels to normal and were not efective as glibenclamide. Ater this, a gradual increase was recorded up to the twenty fourth hour. Intraperitoneal administration of aqueous leaf extract at all four dose levels (50, 100, 200 and 300 mg/kg body weight) of Zanha africana also lowered blood glucose levels from the 1st hour to the 6th hour (Table 2, Figure 2) in a dose independent manner. By the 1st hour, the four extract doses had lowered the blood glucose levels by 6.91%, 10.70%, 13.25%, and 13.70%, respectively, compared to insulin which had lowered blood sugar levels by 68.40% within the same hour. In this hour, all tested dose levels did not lower blood glucose levels to normal. By the 6th hour, all the four dose levels (50, 100, 200 and 300 mg/kg body weight) of Zanha africana lowered blood glucose levels by more than half, that is, 70.22%, 55.49%, 57.65% and 63.56%, respectively, compared to insulin which had lowered blood glucose levels by 76.30% Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 4 of 9 Treatment Normal control Diabetic control Route Glucose levels at varying times in mmole/L 0 hr 1 hr 2 hr 3 hr 4 hr 6 hr 24 hr Oral 5.12 ± 0.15 5.06 ± 0.21 5.06 ± 0.13 5.18 ± 0.13 5.18 ± 0.13 5.04 ± 0.11 5.34 ± 0.22 IP 5.18 ± 0.13 5.08 ± 0.13 5.06 ± 0.15 5.22 ± 0.13 5.26 ± 0.34 5.12 ± 0.13 5.26 ± 0.13 Oral 20.64 ± 1.13A 22.72 ± 1.09Ba 24.32 ± 1.13Ca 24.94 ± 1.07Cb 25.88 ± 0.97Cb 27.84 ± 0.87Cc 29.66 ± 0.76Cd IP 20.52 ± 1.68A 22.98 ± 2.38B 25.34 ± 1.80Ba 26.92 ± 1.57Cb 28.98 ± 1.44Cc 30.30 ± 0.73Cd 30.84 ± 0.39Cd Diabetic/Gliben Oral 21.86 ± 1.98Ad 19.92 ± 2.26Ad 16.50 ± 1.66Ac 12.36 ± 1.47Ab 8.30 ± 0.66a 5.00 ± 0.27 7.46 ± 0.72 Diabetic/Insulin IP 23.04 ± 2.31Aa 7.28 ± 0.66 6.48 ± 0.45 6.00 ± 0.35 5.80±0.51 5.46±0.54 6.84±0.48 Extract dose (mg/kg body weight) 50 100 200 300 Oral 20.28 ± 4.41Aa 12.60 ± 5.56 11.44 ± 3.75 8.74 ± 1.67 8.12 ± 1.23 7.12 ± 1.19 8.22 ± 1.10 IP 23.44 ± 7.38Aa 21.82 ± 7.50Ba 16.68 ± 4.48A 13.44 ± 3.82B 10.88 ± 4.06A 6.98 ± 1.56 10.74 ± 4.51 Oral 26.40 ± 2.49Ab 23.66 ± 2.64Ba 22.90 ± 1.81B 21.50 ± 1.99B 20.06 ± 1.70B 18.54 ± 2.63B A A Aa IP 20.94 ± 7.44 Oral 23.72 ± 5.86Ab 18.70 ± 5.42 B 20.22 ± 5.56Aa 17.02 ± 4.21 16.54 ± 5.57A 12.70 ± 2.90 15.28 ± 5.68A 24.58 ± 3.70Ba 8.92 ± 2.31 9.32 ± 0.85 B 19.44 ± 9.70B 12.18 ± 4.94A 9.54 ± 3.93A 14.90 ± 3.55A IP 18.56 ± 6.37A 16.10 ± 5.79A 14.08 ± 6.05A 11.32 ± 5.83 8.38 ± 3.40 7.86±1.14A 18.98 ± 9.71A Oral 22.48 ± 5.89Aa 19.32 ± 7.27A 16.50 ± 7.16A 14.86 ± 5.76A 11.62 ± 4.13A 9.40±1.74A 14.42 ± 3.99A IP 22.78 ± 3.30Ab 19.66 ± 3.82a 17.10 ± 3.75A 15.12 ± 4.66B 12.84 ± 3.60B 8.30±0.87A 14.96 ± 7.82 Values are expressed as Means ± SD for ive animals per group. Means within respective columns followed by similar upper case letters are not signiicantly different at p ≤ 0.05 by ANOVA and post ANOVA; means within respective rows followed by similar lower case letters are not signiicantly different at p ≤ 0.05 by ANOVA and post ANOVA. Table 2: Effect of oral and intraperitoneal administration of aqueous leaf extracts of Zanha africana on blood glucose levels in alloxan induced diabetic mice. Figure 1: The mean percentage change in blood glucose levels after aqueous leaf extracts of Zanha africana was administered orally in alloxan induced diabetic mice. Normal control % change in blood glucose Diabeic control Diabeic/Insulin 50 100 200 300 Time (hrs) Figure 2: The mean percentage change in blood glucose levels after aqueous leaf extracts of Zanha africana was administered intraperitoneally in alloxan induced diabetic mice. within the same hour. In this hour, the dose level 50 mg/kg body weight decreased blood glucose levels to normal and was efective as insulin while the dose level 200 mg/kg body weight decreased blood glucose levels to normal but was not efective as insulin. However the dose levels 100 mg/kg body weight and 300 mg/kg body weight did not lower blood glucose levels to normal. Ater this, a gradual increase was Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal recorded up to the twenty fourth hour. Efect of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on body and organ weights in mice Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 5 of 9 Table 3 shows the efect of oral and intraperitoneal administration of aqueous leaf extracts of Zanha africana at 1 g/kg body weight to mice for one month on the weekly changes in body weight and percent organ to body weight. Oral and intraperitoneal administration of 1 g/ kg body weight of aqueous leaf extracts of Zanha africana to mice for one month signiicantly decreased the weekly body weight gain relative to that of the normal control mice (Table 3). Oral administration of aqueous leaf extracts of Zanha africana at 1 g/kg body weight to mice for one month did not signiicantly alter the percent organ to body weights of all the studied organs relative to those of the normal control mice (Table 3). In addition, administration of the same intraperitoneal dose of aqueous leaf extracts of Zanha africana to mice for one month signiicantly increased the percent organ to body weight of liver, brain, and kidney but did not signiicantly alter the percent organ to body weight of the lungs, spleen, heart, and testes relative to those of the normal control mice (Table 3). levels of Neutrophils, Eosinophils, Monocytes, and Basophils relative to those of normal control mice (Table 5). Efects of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on biochemical parameters in mice Oral administration of 1g/kg body weight of aqueous leaf extracts of Zanha africana caused a signiicant increase in levels of γ-GT, LDH, and CK while signiicantly decreasing the levels of Urea, ALT, AST, T-BIL, D-BIL and Creatinine relative to that of the normal control mice; however, no signiicant alteration in the levels of ALP and α-AMY by the same extract dose compared to that of the respective normal control group (Table 6 and 7). Intraperitoneal administration of the same dose of aqueous leaf extracts of Zanha africana signiicantly increased the levels of γ-GT, T-BIL and D-BIL while decreasing the levels of AST and Creatinine relative to that of the normal control mice; however, no signiicant alteration on the levels of Urea, ALT, LDH, CK, ALP, and α-AMY by the same extract dose compared to respective normal control group (Table 6 and 7). Efect of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on hematological parameters in mice Quantitative analysis of the phytochemical composition of the aqueous leaf extracts of Zanha africana Results are shown in Table 4. Oral administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana to mice for one month significantly increased the level of MCH and significantly decreased the level of PLT but did not significantly change the levels of RBC, Hb, PCV, MCV, and MCHC relative to those of the normal control mice. In addition, administration of the same intraperitoneal dose of aqueous leaf extracts of Zanha africana, to mice for one month did not significantly change the levels of all the measured hematological parameters relative to those of the normal control mice. he results of quantitative analysis of ive major groups of phytochemical constituents in the aqueous leaf extracts of Zanha Africana are shown in Table 8. Mineral elements analysis Aqueous leaf extracts of Zanha Africana contained Sodium (Na), Chlorine (Cl), Potassium (K), Calcium (Ca), Titanium (Ti), Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Copper (Cu), Zinc (Zn), Arsenic (As), Cadmium (Cd), Magnesium (Mg), Nickel (Ni) and Lead (Pb). he levels of these measured minerals and trace element were all below the recommended daily allowance (Table 9). Efect of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on white blood cell count in mice Discussion Oral administration of aqueous leaf extracts of Zanha africana at 1g/kg body weight to mice for one month did not cause signiicant change to the diferential white blood cell count (Table 5). In addition, Intraperitoneal administration of the same dose of aqueous leaf extracts of Zanha africana to mice for one month signiicantly increased the levels of WBC and Lymphocytes without signiicantly afecting the Treatment he aim of this study was to investigate the in vivo antidiabetic efect and safety of the aqueous leaf extracts of Zanha africana in alloxan induced diabetic mice and normal mice respectively. he alloxan administration resulted in 3 to 4 times increase in blood glucose levels compared to normal control group. Average weekly change in body weight (g) and percent organ to body weight (%) Δweight/week Liver Brain Kidney Lungs Spleen Heart Testes Control Oral 1.705 ± 0.522 7.27 ± 1.12 2.74 ± 0.27 1.78 ± 0.24 1.79 ± 0.21 1.12 ± 0.32 0.53 ± 0.09 0.93 ± 0.16 Zanha africana Oral 0.340 ± 0.495* 7.83 ± 1.36 2.65 ± 0.92 1.87 ± 0.11 2.28 ± 0.92 1.05 ± 0.41 0.62 ± 0.08 0.92 ± 0.12 Control IP 2.155 ± 0.089 5.01 ± 1.26 1.75 ± 0.31 1.39 ± 0.42 1.38 ± 0.54 0.61 ± 0.13 0.51 ± 0.21 0.91 ± 0.22 Zanha africana IP 0.490 ± 0.189* 9.18 ± 1.74* 2.91 ± 0.37* 2.32 ± 0.49* 2.19 ± 0.69 0.93 ± 0.45 0.71 ± 0.13 0.94 ± 0.14 Results are expressed as Mean ± Standard Deviation (SD) for ive animals for each parameter; *p<0.05 is considered statistically signiicant when the mean of the extract treated group is compared to its relevant control group by t-test. Table 3: The effects of oral and intraperitoneal administration of aqueous leaf extract of Zanha africana at 1 g/kg body weight on body and organ weights in mice. Treatment Hematological parameters RBC (x106/ µL) Hb (g/dL) PCV (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (x103/µL) Control Oral 7.61 ± 0.74 9.58 ± 0.73 32.74 ± 2.49 43.12 ± 2.84 12.64 ± 0.96 29.30 ± 0.54 607.40 ± 116.07 Zanha africana Oral 6.69 ± 1.19 10.08 ± 1.05 30.96 ± 5.48 46.26 ± 2.41 14.40 ± 1.06* 31.16 ± 3.21 436.32 ± 57.54* Control IP 6.38 ± 0.67 8.98 ± 0.80 28.66 ± 2.64 45.02 ± 1.44 14.10 ± 0.51 31.36 ± 0.66 352.80 ± 73.32 Zanha africana IP 6.20 ± 0.66 8.80 ± 1.10 28.14 ± 3.25 45.36 ± 1.93 14.20 ± 0.72 31.26 ± 0.61 439.00 ± 96.11 Results are expressed as Mean ± Standard Deviation (SD) for ive animals in each treatment; *p<0.05 is considered signiicant when the mean of the control animals is signiicantly different from that of the extract treated animals by t-test. Table 4: The effects of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on hematological parameters in mice. Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 6 of 9 White blood cell and differential white blood cell count Treatment WBC (x103/µL) Neutrophils (x103/µL) Lymphocytes (x103/µL) Eosinophils (x103/µL) Monocytes (x103/µL) Basophils (x103/µL) Control Oral 14.34 ± 3.48 4.46 ± 1.26 7.27 ± 1.68 1.35 ± 0.41 1.15 ± 0.21 0.09 ± 0.08 Zanha Africana Oral 12.82 ± 6.38 3.50 ± 1.70 7.12 ± 3.64 1.17 ± 0.55 0.95 ± 0.43 0.08 ± 0.10 Control IP 6.87 ± 0.71 2.18 ± 0.25 3.51 ± 0.33 0.59 ± 0.14 0.54 ± 0.13 0.04 ± 0.04 Zanha africana IP 9.08 ± 0.94* 2.44 ± 0.32 5.08 ± 0.46* 0.84 ± 0.13 0.71 ± 0.15 0.04 ± 0.05 Results are expressed as Mean ± Standard Deviation (SD) for ive animals in each treatment; *p 0.05 is considered signiicant when the mean of the control animals is signiicantly different from that of the extract treated animals by t-test. Table 5: The effects of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on white blood cell count (WBC) in mice. Enzyme Activities Treatment ALT (U/L) AST (U/L) GGT (U/L) LDH (U/L) CK (U/L) AMY (U/L) Control Oral 132.6 ± 20.6 692.3 ± 51.4 1.8 ± 0.2 1972.9 ± 158.7 953.4 ± 74.7 2940.2 ± 174.7 ALP (U/L) 103.2 ± 9.1 Zanha africana Oral 66.2 ± 7.5* 356.1 ± 42.2* 3.6 ± 0.9* 2182.2 ± 100.4* 1155.6 ± 96.9* 2798.0 ± 311.8 104.7 ± 9.4 Control IP 80.3 ± 7.0 523.2 ± 94.7 2.0 ± 1.0 2137.2 ± 159.4 351.0 ± 59.1 1676.4 ± 230.2 46.6 ± 10.4 Zanha africana IP 98.8 ± 21.5 383.2 ± 48.4* 4.0 ± 1.2* 2087.2 ± 265.8 336.0 ± 92.4 1650.0 ± 209.3 52.6 ± 20.0 Results are expressed as Mean ± Standard Deviation (SD) for ive animals in each treatment; *p<0.05 is considered signiicant when the mean of the control animals is signiicantly different from that of the extract treated animals by t-test. Table 6: The effects of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on organ functions in mice. Treatment Metabolite Levels UREA (mmole/L) CREAT (µmole/L) T-BIL (µmole/L) Control Oral 9.6 ± 0.7 75.0 ± 8.5 0.8 ± 0.1 D-BIL (µmole/L) 0.4 ± 0.1 Zanha Africana Oral 7.4 ± 0.6* 54.4 ± 3.2* 0.5 ± 0.1* 0.3 ± 0.1* Control IP 7.2 ± 0.8 43.2 ± 6.1 4.5 ± 1.2 2.6 ± 0.7 Zanha africana IP 8.2 ± 0.8 28.6 ± 3.5* 8.7 ± 0.9* 5.3 ± 0.8* Results are expressed as Mean ± Standard Deviation (SD) for ive animals in each treatment; *p<0.05 is considered signiicant when the mean of the control animals is signiicantly different from that of the extract treated animals by t-test. Table 7: The effects of oral and intraperitoneal administration of 1 g/kg body weight of aqueous leaf extracts of Zanha africana on the levels of selected metabolites in mice. Both oral and intraperitoneal route of administration of the aqueous extract of the studied plant showed hypoglycemic activity at the four tested dose levels (50 mg/kg body weight 100 mg/kg body weight, 200 mg/kg body weight, and 300 mg/kg body weight). hese inding agrees with the results obtained by Mukundi et al. [28] who undertaken a similar study and reported that the aqueous leaf extracts of Acacia nilotica showed hypoglycemic activity in alloxan induced diabetes mice. alkaloids obtained from leaves of Acanthus montanus administered intraperitoneally at doses of 100, 200 and 400mg/kg body weight demonstrated hypoglycemic activity in alloxan-induced diabetic rats [35]. Four indole Alkaloids isolated from the leaves of Catharanthus roseus increased glucose uptake in pancreatic and muscle cells. Moreover these alkaloids were found to inhibit protein tyrosine phosphatase PTP-1B which is a down regulator in the insulin signaling pathway [36]. his hypoglycemic activity could be due to the presence of lavonoids, alkaloids, saponins, tannins and total phenols in the studied plant extract (Table 7). Flavonoids are shown to stimulate peripheral glucose uptake and regulate the activity and/or expression of the ratelimiting enzymes involved in carbohydrate metabolism pathway by acting as insulin secretagogues or insulin mimetics [29] Flavonoids isolated from Pterocarpus marsupium has been shown to in vitro cause pancreatic β-cell regranulation and found to enhance insulin release and conversion of proinsulin to insulin [30]. In addition to phytochemical components, the hypoglycemic activity of the studied plant extract could result from its mineral/ trace elements composition. Zinc is required in all aspects of insulin metabolism, synthesis, secretion and utilization. here is a high zinc excretion rate in diabetic patients and zinc supplementation was shown to improve insulin levels in both type 1 and type 2 diabetes [37]. Presence of saponins in this extract could also be responsible for the hypoglycemic activity. For instant total saponins from the seeds of Entada phaseoloides showed signiicant decrease in fasting blood glucose levels in type 2 diabetic rats [31]. In additions saponins were found to reduce serum glucose levels in elderly diabetic patients [32]. It was reported that condensed tannins obtained from some Kenyan foods inhibited of α-amylase and α-glucosidase enzymes [33] in addition, Commercially available tannic acids were shown to induce phosphorylation of the insulin receptor (IR) and cause translocation of glucose transporter 4 (GLUT 4) [34]. Alkaloids are also reported to be antidiabetic. For example Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Magnesium plays important role in glucose transport across cell membranes and is found as a cofactor in various enzymes involved in glucose oxidation pathways [38]. It was demonstrated that 4 weeks dietary Mg supplementation improved insulin secretory capacity [39]. Studies on guinea pigs showed that manganese deiciency caused impaired glucose tolerance and this was corrected by manganese supplementation [40]. In vitro and in vivo studies demonstrated that selenium exhibits insulin like activities, such as glucose uptake stimulation and regulation of metabolic pathways like glycolysis, pentose phosphate pathway, fatty acid synthesis and gluconeogenesis [41]. Experimental studies demonstrated that hypokalemia resulting from use of potassium wasting diuretics caused decrease in the pancreatic capacity to secrete insulin, diminished β-cell sensitivity to insulin and hence caused impaired glucose tolerance [42]. However Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 7 of 9 Phytochemical content Extract Zanha africana Tannins Total Phenols Flavonoids Saponins Alkaloids 0.7671 ± 0.004 1.8503 ± 0.077 1.5830 ± 0.342 52.333 ± 3.960 56.520 ± 7.806 Results are expressed as Mean ± Standard Deviation (SD). Tannins and total phenols are expressed as mg/g gallic acid equivalent (GAE), lavonoids are expressed as mg/g quercetin equivalent (QE), alkaloids and saponins are in mg/g. Table 8: Quantitative analysis of the phytochemical composition of the aqueous leaf extracts of Zanha africana. Element Zanha africana (µg/g) Amount given to RDA for mice (µg/day)* each mouse (µg) Na* 1893.3 ± 128.2 47.3325 5 x 105 (178.6) Mg* 205.9 ± 41.7 5.1475 4.2 x 105 (150) Cl* 143.2 ± 1.9 3.58 7.5 x 105 (267.9) K* 1474.6 ± 13.7 36.865 3.5 x 106 (1250) Ca* 50.8 ± 0.6 1.27 1.0 x 106 (357.1) Ti 0.41 ± 0.05 0.01025 V 0.16 ± 0.03 0.004 Cr* 0.012 ± 0.006 0.0003 3.5 x 10(12.5) Mn* 2.41 ± 0.05 0.06025 2.3 x 103 (0.82) Fe* 16.29 ± 0.19 0.40725 8.0 x 103 (2.9) Ni 0.57 ± 0.02 0.01425 <1 x 103 <(0.36) Cu* 0.23 ± 0.01 0.00575 1.5 x 103 (0.54) Zn* 1.36 ± 0.03 0.034 1.1 x 104 (3.9) As 0.05 ± 0.01 0.00125 Se <0.030 <0.00075 Pb 0.08 ± 0.01 0.002 Cd 7 ± 0.9 0.175 <1.8 x 103 <(0.64) 3.5 x 10(0.0125) Results on the concentration of each mineral are expressed as µg/g of dry powder of the aqueous leaf extracts of Zanha africana of three determinations and are in the second column; the amount of each mineral administered in µg based on its concentration in the plant’s extract is in the third column. This is compared with the recommended daily allowance shown in the last column. This is expressed per the average weight of each mouse. *Recommended daily allowance estimated from that of human beings stated in Strain and Cashman (2009) [60]. Table 9: Mineral levels and amount given to each mouse from the aqueous leaf extracts of Zanha africana potassium supplementation was found to improve insulin sensitivity, responsiveness and secretion [43,44]. he trivalent Cr is a part in biologically active substance called glucose tolerance factor (GTF), that regulates glucose biotransformation and increases the number of insulin receptors, enhances receptor binding, and potentiates insulin action [37,45]. Experimental chromium deiciency was found to leads to impaired glucose tolerance, which is improved by chromium supplementations [38,45]. Calcium improves insulin sensitivity in some type 2 diabetic patients [46]. Vanadium acts as phosphate analog and exerts efects on several steps in the insulin signaling pathways [45]. Animal model studies showed that vanadium enhanced insulin sensitivity and increased glucose uptake [47,48]. he oral and intraperitoneal administration of the aqueous leaf extracts of Zanha africana caused decrease in growth rate. his decrease in growth rate could be due to the presence of alkaloids, saponins, lavonoids, and tannins. For instance, lavonoids decrease body weight through decreasing glucose absorption. his leads to an increase in fat oxidation. Catechins (lavanoids) are reported to reduce body weight possible by two mechanisms: Inhibition of small-intestine micelle formation and inhibition of α-glucosidase activity which would lead to a decrease in carbohydrate absorption [49]. Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Alkaloids which are present in high amounts in the aqueous extracts of Zanha africana are found to cause weight loss. Alkaloids like Synephrines and ρ-octopamine cause a decrease in body weight by increasing resting energy expenditure, energy used in physical activity and thermal efect of feeding, by 70%, 20% and 10% respectively [49]. Nicotine an alkaloid mainly found in tobacco plant has been reported to act on the central nervous system and modulate several pathways that regulate the aspects of food intake leading to reduced appetite. Cathinone (monoamine alkaloid) delays gastric emptying and hence reduces appetite by acting on the hypothalamus [49]. For the extract administered through the oral route, tannins are reported to reduce feed intake by decreasing palatability and by reducing feed digestion. Palatability is reduced because astringency efect of tannins. Astringency is the sensation resulting from formation of complexes between tannins and salivary glycoproteins. Reduced palatability depresses feed intake. Reduction in digestibility negatively afects intake by causing illing efect due to presence of undigested food [50]. Protanthocyanidins which are condensed tannins cause damage to the mucosa of the gastrointestinal tract resulting in decreased absorption of nutrients. They are also found to increase excretion of proteins and essential amino acids [51]. Mineral element overdose may also cause toxicity but this was not the case with the measured minerals since their levels were below the recommended daily allowance. he increased percent organ to body weight of liver, brain and kidneys of mice intraperitoneally administered with the aqueous leaf extracts of Zanha africana at 1 g/kg body weight daily for one month could not be explained in this study. It is possible that the extract promoted higher metabolic activity in these organs. he investigated hematological parameters in this study are important in the assessment of the potential toxic efect of the plant extract on the bone marrow activity and hemolysis [52]. he main reason for measuring RBC is to check anemia and to evaluate erythropoiesis. Hemoglobin level indicates oxygen carrying capacity of the body, while packed cell volume helps to determine the degree of anemia or polycythaemia. he mean cell hemoglobin level is an important index for folic acid and or vitamin B12 need. Platelets are important for blood clotting, they initiate repair of blood vessels walls and act as an acute phase reactant to infection or inlammation [53]. Studies have shown that use of medicines or herbal drugs can alter the normal range of hematological parameters [54]. In the present study the oral administration of aqueous leaf extracts of Zanha africana caused an increase in MCH levels and decreased platelet count. his may be due to the toxic constituents in this plant extracting including; total phenols, alkaloids, saponins, lavonoids, and tannins present in this plant extracts. Saponins hemolyse and cause cell death in many tissues [55,56]. Alkaloids have been shown to cause liver megalocytosis, proliferation of biliary tract epithelium, liver cirrhosis and nodular hyperplasia [57]. his toxicity may not have been due to the presence of trace elements/ Volume 6 • Issue 9 • 1000422 Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. Pharm Anal Acta 6: 422. doi:10.4172/21532435.1000422 Page 8 of 9 minerals since the amounts administered into each mouse daily at a dose of 1g/kg body weight were below the recommended daily allowance. he signiicant increase in white blood cells observed on intraperitoneal administration of plant extracts of Z. africana indicates a more accelerated production of these cells and a boosted immunity to mice treated by this extract [58-60]. his could be due to tissue damage caused by some constituents of this plant extract. his argument is in line with the observed enlargement of the liver, brain and kidney and the altered levels of alanine transaminase, aspartate aminotransferase, γ-glutamyl transpeptidase, lactate dehydrogenase, creatine kinase, urea, creatinine, total bilirubin and direct bilirubin in mice administered with 1g of Z africana extracts/ kg body weight. he observed signiicant increase in lymphocytes (main efectors cells of the immune system) on intraperitoneal administration of aqueous extracts of Z. africana at 1 g/kg body weight in mice for 28 days indicates a possible stimulatory efect by this extract on lymphocyte production. Conclusion he aqueous leaf extracts of Zanha africana had antidiabetic activity. he aqueous extract of the studied plant at high dose of 1 g/ kg body weights which is far from the therapeutic dose tends to cause toxicological efects. his was well demonstrated in the body and organ weight changes, hematological, and biochemical parameters. In the toxicological studies the oral administration of the high dose (1 g/kg body weight) was found to have less toxic efects than the intraperitoneal administration of the same dose. his explains why the oral route is the most preferred route by the traditional health practitioners. he antidiabetic and toxic action of the studied plant may have resulted from its phytochemical and mineral constituents. Kenya. National Museums of Kenya. 12. Kambizi L, Afolayan AJ (2001) An ethnobotanical study of plants used for the treatment of sexually transmitted diseases (njovhera) in Guruve District, Zimbabwe. J Ethnopharmacol 77: 5-9. 13. 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Halberstam M, Cohen N, Shlimovich P, Rossetti L, Shamoon H (1996) Oral vanadyl sulfate improves insulin sensitivity in NIDDM but not in obese nondiabetic subjects. Diabetes 45: 659-666. OMICS International: Publication Benefits & Features Unique features: • • • Increased global visibility of articles through worldwide distribution and indexing Showcasing recent research output in a timely and updated manner Special issues on the current trends of scientiic research Special features: Citation: Abdirahman YA, Juma KK, Makori WA, Agyirifo DS, Ngugi MP, et al. (2015) Blood Glucose Lowering Effect and Safety of the Aqueous Leaf Extracts of Zanha africana. 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