DE GRUYTER Journal of Complementary and Integrative Medicine. 2018; 20170139 Jeremiah Oshiomame Unuofin1 / Gloria Aderonke Otunola1 / Anthony Jide Afolayan1 Acute and subacute toxicity of aqueous extract of the tuber of Kedrostis africana (L.) Cogn in Wistar rats 1 Medicinal Plants and Economic Development (MPED) Research Centre, Department of Botany, University of Fort Hare, Alice 5700, South Africa, E-mail: unuofinjeremiah@gmail.com, gotunola@ufh.ac.za, aafolayan@ufh.ac.za Abstract: Kedrostis africana (L.) Cogn (Cucurbitaceae) is used in South African traditional medicine and pharmacopoeia as an emetic, purgative and diuretic, and it is used against dropsy in the management of obesity. Aim of the study: In this study, acute and subacute toxicity of aqueous extract of K. africanatuber was evaluated in male and female Wistar rats in order to assess its safety profile. Materials and methods: In acute toxicity, the effects of a single oral dose (2,000 and 5,000 mg/kg) of aqueous extract was determined in both sexes. General behavior, adverse effects and mortality were determined for 3 h and then periodically for 14 days. The subchronic toxicity test was performed in rats. The effects of the extract in daily single oral administration at the doses of 200, 400 and 600 mg/kg for 28 days were determined. Food and water intakes were monitored daily while body weight was monitored on a weekly bases. Hematological, biochemical and organ parameters were determined at the end of the 28-day administration. Results: In the acute study, a single administration of the aqueous extract at the doses of 2,000 and 5,000 mg/kg did not induce mortality. Thus, the LD50 of the aqueous extract of K. africana (AEKA) has been estimated to be higher than 5,000 mg/kg. In the subchronic study, daily oral administration of the AEKA did not result in death of the rats or significant changes in hematological or biochemical parameters at the highest dose of 600 mg/kg. No alteration was observed in body weight, food and water intake. Liver, kidney and heart histopathology did not reveal morphological alteration. Conclusions: The results showed that the aqueous tuber extract of K. africana did not cause any death, nor did it cause abnormalities in necropsy and histopathology findings. There were no acute or subchronic toxicity observed, and this indicates that the plant extract could be considered safe for oral medication. Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Keywords: acute toxicity, biochemical parameters, hematological parameters, Kedrostis africana, subacute toxicity DOI: 10.1515/jcim-2017-0139 Received: November 11, 2017; Accepted: April 14, 2018 Introduction The reliance on plants for food and medicines by mankind is dated back to ancient time, and this practice is still being appreciated in modern times. According to the WHO report, about 80% of developing country population depend on traditional medicine for their primary health care [1, 2]. This could be attributed to the scarcity and high costs of available drugs [3] and also the easy access to these plants in some regions of the world, particularly Africa, Asia and South America. Traditional medicine and medicinal plants have been frequently used in urban settings as alternatives in daily health care and also for self-medication against minor and chronic ailments in less wealthy rural areas [4]. The use of herbal remedies has tremendously gained increasing patronage even in developed countries. The main reason for this increased patronage could be attributed to the belief of their efficacy and safety. The false assertion about their origin however should not guarantee their safety, since there are no sufficient preclinical, safety and animal toxicity report to support the nutritional or beneficial claims made for many of these herbal products [5]. Nevertheless, research has shown that many plants used as food or in traditional medicine are potentially toxic, mutagenic and carcinogenic [6, 7]. According to Adewunmi and Ojewole [8], the potentially toxic and lethal constituents in traditional medicinal products include pyrrolizidine alkaloids, benzophenanthrine alkaloids, lectins, saponins, diterpenes, cyanogenic glycosides and furanocoumarins. It is therefore necessary to evaluate the safety of herbal remedies, especially to Gloria Aderonke Otunola is the corresponding author. © 2018 Walter de Gruyter GmbH, Berlin/Boston. Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 1 Unuofin et al. DE GRUYTER determine the consequence of their prolonged use, which is critical to the discovery and development of standardized herbal remedies and the acceptance of such by stakeholders in health care for therapeutic application. Kedrostis africana (L.) Cogn belongs to the family Cucurbitaceae. It is endemic to Nambia and South Africa (Eastern Cape, Free State, Gauteng, Kwazulu-Natal, Limpopo, Mpumalanga, Northern Cape, North West and Western Cape). This tuberous plant has a water-storage organ, thus making it resistant to drought [9]. Unuofin and co-workers reported the tuber of K. africana contains 6.95% protein and 1.12% crude fat, expressed in percent of dry matter. The ash content was also found to be 16.28%, and mineral compositions, such as calcium 2,505 mg/100 g, magnesium 485 mg/100 g, potassium 2,225 mg/100 g, phosphorus 240 mg/100 g, sodium 430 mg/100 g, zinc 4.80 mg/100 g, copper 0.10 mg/100 g, manganese 3.10 mg/100 g and iron 89.9 mg/100 g, were reported [10]. In Khoi-San and Cape Dutch medicine, the tuber is used as an emetic, purgative and diuretic, and it is used against dropsy [11]. In the Eastern Cape of South Africa, the crushed fresh bulb is used for the management of obesity folkolrically [12, 13]. Biological and pharmacological activities attributed to different solvent extracts of K. africana are as diverse as antioxidant, antibacterial, fungicidal and larvicidal activities [14, 15]. Regarding the ethnobotanical informations and different activities reported with K. africana, it becomes necessary to evaluate the toxic effects of the aqueous extract of the plant that might occur with the administration of single or repeated doses. Materials and methods Location and collection of sample K. africana used for this study was collected in August 2015 in Fort Beaufort in the Amathole District Municipality, Eastern Cape, South Africa. This area lies at latitude 32°43฀,28.66„and longitude 26°34฀5.88฀฀. The plant was authenticated by Mr Tony Dold of Selmar Schonland Herbarium, Rhodes University, South Africa, and a voucher specimen (Unuofin Med, 2015/2) was prepared and deposited in the Giffen Herbarium, University of Fort Hare. Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Preparation of extract The bulb was rinsed, chopped into small bits, oven dried (LABOTEC, South Africa) at 40 °C for 72 h and then ground into powder (Polymix® PX-MFC 90D Switzerland). The ground sample (200 g) was weighed into conical flasks containing 2 L water and shaken in an orbital shaker (Orbital Incubator Shaker, Gallenkamp) for 48 h. The crude extracts were filtered under pressure using a Buchner funnel and Whatman No. 1 filter paper. The filtrate obtained was concentrated using a freeze dryer (Vir Tis benchtop K, Vir Tis Co., Gardiner, NY, USA). The extract was administered orally, using gavage at the respective doses for the acute and subacute toxicity studies. Experimental animals Healthy albino rats of the Wistar strain (both sexes) weighing 114±17 g were purchased from the South African Vaccine Producers, Johannesburg, South Africa, and were used for the studies. They were housed in the animal house of the Central Analytical Laboratory, University of Fort Hare Alice, 5700, South Africa. The rats were kept under standard laboratory conditions (12 h light and dark cycle; 22±2 °C). The animals were fed with standard rat pellet diet and water ad libitum. The study was carried out and the animals were handled according to the guidelines of the National Research Council: Guide for the Care and Use of Laboratory Animals, Committee on Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources DHHS (NIH Publication No. 1985:85–93). The study was approved by the University of Fort Hare Animal Use Research Ethics Committee, South Africa with protocol number AFO051SUNU01. Acute oral toxicity study The acute toxicity study was conducted following the OECD guidelines 420 [16]. Eighteen (18) animals, including nine females and nine males, were used. The predetermined dose of the extract was the selected 2 Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM DE GRUYTER Unuofin et al. doses of 2,000 mg/kg of body weight, which was administered first in three females then repeated with three males. The dose 5,000 mg/kg was administered thereafter; according to the results obtained at a single dose of 2,000 mg/kg. The animals were maintained on a standard animal diet and water. Treated groups were observed for general behavioral changes, symptoms of toxicity, changes in body weight, and mortality after treatment for the first 4 h, then over a period of 24 h, and thereafter daily for 14 days. The rats were fasted for 16–18 h and then sacrificed. The liver, kidney, heart, lung and pancreas were excised, weighed and observed for any histopathological defects. Subacute oral toxicity study This study was conducted in accordance with the Organization for Economic Cooperation and Development (OECD) Test Guidelines 407 with slight modifications [17]. Forty animals weighing 114±17 g were randomly divided into four groups of 10 rats each, consisting of five males and females. Three groups of 10 rats were daily treated by oral administration of the plant extract at different doses of 200, 400 and 600 mg/kg for 28 days. The control group consisted of five males and five females who were also treated for 28 days with distilled water at the dose of 10 mL/kg. All animals were treated daily for 28 days and the animals were observed twice daily for any adverse effect or toxic signs and behavioral changes, mortality and morbidity till the completion of the experiment. The aqueous was orally administered to each group of rats daily for 28 days, while the control group received the water vehicle. Rats were fasted overnight, anesthetized using diethyl ether and sacrificed after the 29th day. Body weights of the rats in all groups were recorded before the start of dosing once weekly during the treatment period and finally on the days of sacrifice. Food and water intake were recorded daily. Paired blood samples, heparinized and nonheparinized, were collected for hematological and serum biochemical assays. Relative organ weight Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd On the 29th day, the animals were sacrificed following an overnight fast. Organs such as liver, lungs, kidneys, heart and spleen were carefully dissected out and weighed in grams. The relative organ weight of each animal was calculated as follows: Relative organ weight=absolute organ weight (g) × 100/body weight of rats on sacrifice day (g) Blood sampling Blood samples were collected via cardiac puncture technique. Blood was divided into two parts: one part was collected in plain bottles (nonheparinized), while the second part was collected in heparinized bottles. The blood samples were subsequently centrifuged at 3,000 rpm for 10 min using a bench centrifuge (Hermle Z 306, Labortechnik GmbH, Germany) to obtain serum and plasma, respectively. The serum and plasma obtained was separated and transferred into fresh plain sample bottles and used for the subsequent hematological and biochemical analyses. Hematological analyses The heparinized blood was analyzed for white blood cell (WBC) count, red blood cell (RBC) count, differential leukocyte counts, red cell distribution width (RCDW), platelet count, hematocrit, hemoglobin estimation (HB), mean cell volume (MCV), mean corpuscular hemoglobin (MCH) mean corpuscular hemoglobin concentration (MCHC), neutrophils, lymphocytes, monocytes and eosinophils [18, 19]. Biochemical analyses Biochemical analyses carried out include measurement of activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase (ƔGT) activities. Other assays include total protein, calcium, magnesium, chloride, glucose and albumin to assess the liver function, serum total protein, urea, uric acid, creatinine, total bilirubin (Total bil) and conjugated bilirubin (Con Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 3 DE GRUYTER Unuofin et al. bil) concentrations for kidney function. Also, the concentration of serum triglyceride and total cholesterol was also determined to give an indication of the effects of these extracts on lipid profile. Histopathological studies The liver and kidney excised from each group of the animals were subjected to histopathological examinations. After fixing the tissues in 10% formalin, they were dehydrated and mounted in paraffin blocks. Sectioning was done at 5–7 μM. Routine histopathology was performed using the hematoxylin stain. Data analysis The statistical analysis was done on MINITAB version 17 for Windows. Data were expressed as mean±SD of five replicates and were subjected to one-way analysis of variance (ANOVA) followed by Fischer’s least significant difference to determine significant differences in all the parameters. Values were considered statistically significant at p < 0.05. Results Acute toxicity Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd At graded doses of 2,000 and 5,000 mg/kg body weight of aqueous extract of K. africana (AEKA), animals (both sexes) did not show any signs of adverse reactions and no changes in animals’ behaviors during daily monitoring up to 14 days after the administration of the extract. No significant difference in body weight gain was observed between the control and extract-administered groups after 24 h, 7 days and 14 days. In addition, no mortality was recorded throughout the period of observation. As there were no mortality and clinical signs of toxicity in all the tested doses, LD50 value of AEKA tuber was found to be greater than 5,000 mg/kg (data not shown). Subacute toxicity Effects of the aqueous extract of K. africanatuber on body weights Oral administration of AEKA tubers at doses of 200, 400 and 600 mg/kg body weight for 28 days did not produce any mortality in tested animals. There was no significant difference in weights of K. africana treated rats (both sexes) in comparison with the control groups (Table 1). Table 1: Body weights of female and male rats following 28-day subacute oral administration of different doses of K. africana at different concentrations. Body weight, g/week 4 Female 0 1 2 3 4 Control 200 mg/kg 400 mg/kg 600 mg/kg Male Control 200 mg/kg 400 mg/kg 600 mg/kg 102.05±7.08a 99.92±4.46a 95.98±0.98a 96.98±1.81a 129.99±9.50a 123.18±2.75a 121.59±1.21a 124.71±4.39a 163.32±7.41a 158.73±2.70a 158.03±2.10a 159.18±3.23a 169.60±9.89a 164.53±4.78a 168.56±8.81a 167.63±7.82a 187.03±6.96a 187.46±7.39a 185.25±5.18a 186.24±6.12a 114.12±6.47a 109.70±3.07a 107.38±0.74a 110.32±3.68a 152.47±11.94a 149.57±9.06a 147.66±6.98a 144.37±3.84a 213.40±23.39a 194.16±4.23a 197.21±7.26a 199.45±9.49a 241.73±12.79a 238.78±9.87a 237.44±8.58a 236.06±7.17a 281.10±10.53a 276.49±5.96a 273.25±2.71a 278.58±8.03a Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM DE GRUYTER Unuofin et al. Values are expressed as mean± SD, n=5 (females or males). Columns with the same letter are not significantly different from control group (p > 0.05). Effects of the aqueous extract of K. africana tuber on food and water intakes During dosing (28-day) period, there was no significant change in food and water intake in both the female and male rats at all the doses of AEKA tested as compared to their respective control groups (Table 2). Table 2: Effect of the aqueous extract of K. africana on food and water intakes in subacute toxicity. Treatment Sex Average food intake (g/day/rat) Average water intake (mL/day/rat) Control Female Male Female Male Female Male Female Male 12.73 + 1.64a 18.34 + 1.39a 12.09 + 0.99a 16.94 + 0.01a 11.55 + 0.45a 16.97 + 0.03a 11.73 + 0.65a 16.96 + 0.02a 18.74 + 3.94a 24.19 + 4.86a 16.07 + 1.26a 21.71 + 2.59a 16.32 + 1.48a 22.39 + 3.29a 17.28 + 2.51a 22.12 + 2.98a 200 mg/kg 400 mg/kg 600 mg/kg Values are expressed as mean±SD, n=5 (females or males). Columns with the same letter are not significantly different from control group (p > 0.05). Effect of the aqueous extract of K. africana tuber on organ weights Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd There is the likelihood that the ingestion of herbal products into the body could be toxic to important organs such as the liver, kidney, spleen, heart and lungs because of the crucial roles they play in the body. However, there was no significant difference in organ weights of K. Africana-treated rats (both sexes) as compared to the control rats (Table 3). Table 3: Relative organ weights (per 100 g body weight) of rats in subacute toxicity of aqueous extracts K. africana. Relative organ weight (g) Group Male Heart Liver Lungs Kidney Spleen Female Heart Liver Lungs Control 200 mg/kg 400 mg/kg 600 mg/kg 0.96±0.03a 0.96±0.05a 0.90±0.00a 0.98±0.06a 9.11±3.34a 9.08±3.32a 9.09±3.27a 9.12±3.33a 1.11±0.39a 1.12±0.36a 1.13±0.39a 1.14±0.41a 2.02±0.72a 2.08±0.79a 2.02±0.71a 2.07±0.76a 0.60±0.21a 0.62±0.24a 0.59±0.21a 0.59±0.21a 0.61±0.33a 0.6±0.36a 0.60±0.36a 0.59±0.33a 6.04±3.23a 6.03±3.13a 5.63±3.17a 5.36±3.04a 1.01±0.54a 1.35±0.71a 0.47±0.25a 1.04±0.56a 1.37±0.70a 0.47±0.26a 1.02±0.52a 1.36±0.71a 0.48±0.25a 1.05±0.59a 1.36±0.72a 0.46±0.26a Kidney Spleen Values are expressed as mean±SD, n=5 (females or males). Columns with the same letter are not significantly different from control group (p > 0.05). Effects of the extract of K. africana on hematological parameters The effect of daily administration of the aqueous extracts of K. africana (AEKA) on different hematological parameters, namely WBC, RBC, Hem, MCV, MCH, MCHC, RDW, MPV platelets, neutrophil, lymphocyte, basophils, hematocrit, eosinophil and monocyte, did not show any significant differences (p > 0.05) between the experimental and control groups at all the tested doses for both sexes (Table 4). Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 5 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd 6 Male Control 200 mg/kg 400 mg/kg 600 mg/kg Female Control 200 mg/kg 400 mg/kg 600 mg/Kg WBC (×109 /L) RBC (×1012 /L) Hem (g/dL) MCV (fL) MCH (pg) MCHC (g/dL) RDW (%) Platelet count (×109 / L) MPV (fL) Neutrophils (×109 /L) Lymphocytes (×109 /L) Monocytes (×109 /L) Eosinophils (×109 /L) Basophils (×109 /L) Hematocrit (L/L) 10.14±0.74a 7.74±0.30a 14.97±0.31a 64.93±0.79a 19.3±0.36a 29.73±0.26a 11.5±0.14a 781±65.31a 10.37±0.99a 8.43±0.93a 15.23±0.59a 67.57±3.59a 19.49±0.48a 30.9±1.29a 11.57±0.21a 1,033.33±317.69a 10.25±0.85a 8.09±0.69a 15.1±0.49a 65.25±1.28a 19.31±0.39a 30.32±0.85a 11.53±0.18a 923.83±208.14a 11.75±2.35a 8.61±1.12a 16.4±1.78a 66.8±2.74a 19.37±0.42a 31.4±1.95a 11.63±0.28a 764.67±49.77a 7.42±1.65a 8.15±0.14a 14.97±0.09a 63±1.04a 18.43±0.29a 29.23±0.26a 10.87±0.81a 778.67±92.32a 9.9±4.14a 8.28±0.26a 14.99±0.06a 67.83±5.83a 18.63±0.46a 32.97±3.97a 10.57±0.29a 807±118.85a 7.02±1.19a 8.72±0.69a 15.09±0.24a 69.6±9.67a 18.9±0.79a 31.73±3.86a 10.3±0.14a 909.33±224.68a 8.29±2.59a 8.37±0.38a 15.03±0.09a 69.1±9.63a 18.9±0.82a 31.57±3.68a 10.4±0.35a 750.67±64.65a 9.97±0.05 a 0.41±0.10a 9.99±0.07a 0.38±0.08a 9.92±0.01a 0.30±0.01a 9.97±0.09a 0.42±0.13a 9.27±0.31a 0.64±0.06a 8.98±0.07a 0.79±0.24a 8.93±0.01a 0.74±0.19a 8.96±0.05a 0.62±0.03a 6.32±0.90a 6.63±1.25a 6.48±1.06a 5.82±0.41a 4.84±0.94a 4.90±0.90a 6.85±2.96a 7.00±3.10a 2.09±0.12a 1.98±0.01a 1.98±0.00a 2.05±0.19a 1.34±0.53a 1.46±0.61a 1.6±0.75a 1.53±0.71a 0.09±0.04a 0.13±0.07a 0.11±0.06a 0.11±0.05a 0.09±0.05a 0.15±0.10a 0.18±0.12a 0.11±0.05a 0.04±0.00a 0.03±0.00a 0.03±0.00a 0.23±0.20a 0.02±0.00a 0.03±0.00a 0.03±0.00a 0.03±0.00a 0.50±0.01a 0.49±0.00a 0.50±0.00a 0.52±0.01a 0.51±0.00a 0.50±0.00a 0.5±0.00a 0.50±0.01a Values are expressed as mean±SD, n=5 (females or males). Columns with the same letter are not significantly different from control group (p > 0.05). DE GRUYTER Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM Parameter Unuofin et al. Table 4: Effects of aqueous extract of K. africana on hematological parameters. DE GRUYTER Unuofin et al. Effect of the extract on biochemical parameters of rats after 28 days of treatment Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd The biochemical parameters (Table 5) are indicator of major toxic effects in tissues such as kidney and liver. A number of enzymes and proteins can be used to indicate hepatocellular effects (such as ALT, AST, ƔGT and bilirubin). Levels of cholesterol are an indirect indicator of liver function, whilst the levels of creatinine, uric acid and blood urea nitrogen act as biomarkers of nephron functional injury [20]. All the biochemical parameters tested did not show any significant differences (p > 0.05) between the experimental and control groups at all the tested doses for both sexes (Table 5). Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 7 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd 8 Male Control 200 mg/kg 400 mg/kg 600 mg/kg Female Control 200 mg/kg 400 mg/kg 600 mg/kg Sodium (mmol/L) Chloride (mmol/L) Urea (mmol/L) Creatinine (umol/L) Glucose Calcium (mmol/L) Magnesium (mmol/L) Uric acid (mmol/L) Total Protein (g/L) Albumin (g/L) Total bil (umol/L) Con bil (umol/L) ALT (U/L) AST (U/L) ALP (U/L) Total chol (mmol/L) TAG (mmol/L) HDL chol (mmol/L) CRP (U/L) GGT (U/L) 138.8±0.74a 138.2±0.16a 138.8±0.74a 139±0.79a 140.2±1.72a 139.4±0.92a 138.9±0.43a 138.6±0.12a 103±1.10a 103.8±1.93a 104.8±2.87a 103±1.09a 107.4±3.2a 105.2±0.98a 104.6±0.40a 105±0.79a 4.58±0.28a 5.48±1.18a 6.14±1.84b 5.56±1.32a 5.38±0.66a 5.98±1.23a 6.4±1.64a 6.96±2.22a 30.2±3.37a 27±0.16a 27.8±0.98a 27.4±0.59a 37.8±5.04a 33.6±0.82a 35±2.24a 38±5.23a 5.3±0.38a 2.47±0.03a 5.02±0.08a 2.47±0.03a 4.9±0.17a 2.48±0.05a 4.86±0.16a 2.54±0.09a 5.6±0.58a 2.27±0.21a 5.33±0.35a 2.40±0.35a 5.78±0.73a 2.49±0.45a 5.65±0.62a 2.47±0.42a 0.97±0.06a 1.08±0.19a 1.02±0.08a 1.06±0.16a 1.09±0.05a 1.02±0.00a 1.11±0.08a 1.04±0.01a 0.13±0.02a 0.19±0.07a 0.17±0.05a 0.16±0.03a 0.28±0.05a 0.27±0.03a 0.29±0.06a 0.28±0.05a 52.2±1.83a 52±1.63a 52±1.61a 51.6±1.25a 48.2±2.14a 50±3.96a 51.2±5.14a 50.8±4.74a 17.8±0.4a 14.8±3.71a 18.2±0.75a 17.2±6.09a 18±0.63a 12.4±1.28a 17.6±0.20a 12.8±1.68a 20±0.93a 24±6.36a 19.4±0.05a 19±1.38a 19.8±0.41a 22.2±4.58a 19.52±0.14a 26.4±8.76a 5.4±2.15a 7.60±4.35a 6.20±2.98a 5.6±2.38a 8.6±1.62a 7±0.03a 7.98±1.00a 8.49±1.51a 52.8±5.04a 159.6±20.48a 253.4±13.46a 1.06±0.03a 66.2±18.44a 178.6±39.43a 245.8±5.79a 1.18±0.15a 78±30.19a 177.6±38.52a 251.6±11.67a 1.12±0.12a 72.4±24.60a 176±36.84a 257.6±17.62a 1.2±0.16a 74.2±11.51a 226.8±43.07a 152.8±25.21a 0.93±0.11a 70.6±7.92a 222.8±39.07a 140.6±12.98a 1.09±0.24a 74.6±11.89a 290.4±106.65a 152±24.39a 1.06±0.24a 76.8±14.10a 271.4±87.65a 149.6±22.03a 1.04±0.22a 2.14±0.42a 0.75±0.03a 1.76±0.02a 0.81±0.07a 1.81±0.07a 0.82±0.09a 2.15±0.42a 0.80±0.07a 1.66±0.57a 0.80±0.04a 1.58±0.46a 0.79±0.08a 1.76±0.68a 0.82±0.04a 1.94±0.85a 0.81±0.09a <1 <5 <1 <5 <1 <5 <1 <5 <1 <5 <1 <5 <1 <5 <1 <5 Values are expressed as mean±SD, n=5 (females or males). Columns with the same letter are not significantly different from control group (p > 0.05). DE GRUYTER Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM Parameter Unuofin et al. Table 5: Effect of daily administration of aqueous extracts of K. africana tubers for 28 days on biochemical profiles on both sexes of control and treated rats in subacute toxicity study. DE GRUYTER Unuofin et al. Histopathology Histopathological assessment of organs is often used to provide supportive evidence for hematological and biochemical observations in toxicity studies. The light microscopic examination of major organs (liver, kidney and heart) from the rats (both sexes) on 600 mg/kg body weight/day and those on vehicle control is shown in Figure 1. In this study, none of the organs of the rats at 600 mg/kg body weight/day showed any morphological alterations, abnormalities, specific changes or lesions under the light microscope, suggesting that the rats at lower dose levels than 600 mg/kg body weight/day were also not affected biologically by the aqueous extract of the tuber of K. africana. Figure 1: Microscopic examination of various organs in rats given sterile water and the aqueous extract of the tuber of K. africana for 28 days. (a)~(d): liver; (e)~(h): kidney; (i)~(l): heart. Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Discussion For centuries, medicinal plants have gained attention as an alternative source of medicine for the treatment of a myriad of diseases worldwide. However, there is a dearth of information on the toxicological profile and possible side effects of majority of these medicinal plants [21]. Generally, in order to ascertain the safety profile of medicinal plants, acute and subacute toxicity tests are carried out using laboratory animals (e. g. rodents and nonhuman primatives) [6]. In the present study, we investigated the acute and subacute oral toxicity of aqueous extract of the tuber of K. africana in Wistar rats. The acute toxicity study showed that oral administration of aqueous extract of the tuber of K. africana of 2,000 and 5,000 mg/kg body weight did not induce any lethal effect on the rats. In accordance to 2001 OECD guidelines 423 of acute toxicity, the AEKA can be categorized as category 5 GHS (Globally Harmonized System for chemical classification substances and mixtures) since the highest dose of 5,000 mg/kg body weight did not cause death of the animals [22]. Therefore, it could be suggested that aqueous extract of the tuber of K. africana is practically nontoxic via oral route. In the subacute treatment, administration of the extract at the doses of 200, 400 and 600 mg/kg in both female and male rats did not cause significant changes in food and water consumption as well as the body weight during 28-day oral administration of aqueous extract of the tuber of K. africana (Table 1 and 2). The result indicates that the animals had a healthy growth pattern due to the absence of growth inhibition during this course of repeated doses of aqueous extract of the tubers of K. africana. According to Feres et al. [23], animals that lose 10 % of their initial body weight possibly will not survive, which is an indication of adverse side effects. Similarly changes in organ weights have been used as toxicity indices in animals which could be evaluated in toxicological studies [24–26]. Our findings suggest that the three doses of the aqueous extract of the tuber are nontoxic to all vital organs (liver, kidney, heart, spleen and lungs of both sexes) tested in this study (Table 3). Therefore, this tuber extract is considered safe for maintaining the normal function of the organs. It is extremely important to assay for hematological parameters because the hematopoietic system is one of the most susceptible targets for toxic compounds. It is also used to measure the physiological and pathological status of animals and humans [27]. The main channel for the transport of food nutrients and foreign bodies in the body is blood, and as such its components such as RBCs, white blood counts, platelets and hemoglobin are majorly exposed to greater dosage of toxic compounds. The resulting effects of the damages that occur to the blood cells bring about the compromise of the immune system. Since our findings suggest that three doses of the AEKA did not cause any significant change in the hematological parameters measured as compared to the controls in both sexes (Table 4); it could therefore be inferred that the plant extract is nontoxic. According to Arneson and Brickell [28], transaminases (ALT, AST and Gamma-glutamyl transferase (GGT)) are good excellent biomarkers for liver damage. ALP is often measured in order to estimate the extent to bile duct obstruction [29, 30]. According to Uddin et al. [31], elevated levels of serum transaminase enzyme activities are clear indications of hepatic impairment in animals. In this study, there were no deleterious changes found Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 9 Unuofin et al. DE GRUYTER in the levels of transaminases in the plasma of treated animals. The results of the biochemical studies showed that there were no significant changes in the activities of serum ALT, GGT, AST and ALP at treatment doses of K. africana on both sexes in comparison with their respective control groups (Table 5). The administration of K. africana on both sexes of rats produced a no significant difference in the levels of serum total protein, albumin, total bilirubin, conjugated bilirubin, urea and creatinine in both females and males when compared with their respective control group. According to Johnson et al. [32], biomarkers of kidney function are urea and uric acid, and their bioavailability in the body is an indicator of renal damage. Renal damage is usually marked by an increase in levels of creatinine [33]. The observed result in this study also suggests that the aqueous extract of the plant does not have a negative effect, but rather seems to have a protective effect on the kidney. This may probably be an indication that the extract did not interfere with the capacity of the kidney to excrete these metabolites. It is therefore evident that the extract at doses tested did not cause renal impairment or kidney damage. Hence, this tuber extract is considered safe and has no destructive effect on normal kidney functions. Electrolyte functions are often considered when assessing kidney function. Increase or decrease in electrolytes such as sodium, potassium, chloride and magnesium ions could indicate renal injury [34]. In this study, there was no significant difference in the serum levels of sodium, chloride, glucose, calcium and magnesium in both female and male rats treated with K. africana at all doses tested when compared with their respective control groups (Table 5). Thus, it suggests that the plant did not cause electrolyte imbalance. The administration of the aqueous extracts of K. africana on both sexes of rats produced no significant difference in the levels of triglyceride, high-density level cholesterol and c-reactive protein at all doses tested when compared with their respective control groups (Table 5). Finally, there was no significant difference in the serum level of total cholesterol in both female and male rats treated with K. africana at all doses tested when compared with their respective control groups (Table 5). Microscopic analysis in all tested doses of the aqueous extracts of K. africana showed no changes in vital organs of the treated animals when compared with the control group. Thus, it can be suggested that the extract did not have any toxic effects (Figure 1). Examples of such toxic signs would be characterized by congestion, leukocyte infiltration, degeneration, necrosis, apoptosis and fibrosis in the organ tissues analyzed histologically [35]. Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Conclusions The results of this study suggested that the aqueous extract of the tuber of K. africana up to the dose of 600 mg/kg body weight is relatively safe when administered orally to female and male rats. This can be deduced from the fact that the extract at the different doses did not show any lethality, death or adverse effects on the rats, and did not induce significant alterations in all the biochemical, hematological and morphological parameters investigated in this study, regardless of gender. This could be an assurance for the medicinal use of K. africana in folk medicine. Further investigation on its medicinal and therapeutic efficacy could also be considered. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: The authors wish to appreciate the financial support of Govan Mbeki Research Development Centre (GMRDC), University of Fort Hare, South Africa. Grant number C127. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. References [1] World Health Organization. (2003). Traditional medicine. WHO Factsheet No.134. Available online at:http://www.who.int/mediacentre/factsheets/2003/fs134/en/ (Accessed 31 March, 2003). [2] Campbell-Tofte JIA, Mølgaard P, Winther K. Harnessing the potential clinical use of medicinal plants as anti-diabetic agents. Botanics Targets Ther. 2012;2:7–19. [3] Hudaib M, Mohammad M, Bustanji Y, Tayyem R, Yousef M, Aburjaie M, et al. 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In: McPherson RA, Pincus MR, eds. Henry’s clinical diagnosis and management by laboratory methods. 22nd ed. chap 14. Philadelphia, PA: Elsevier Saunders. 2011. [34] Gowda S, Desai PB, Kulkarni SS, Hull VV, Math AAK, Vernekar SN. Markers of renal function tests. N Am J Med Sci. 2010;2:170–73. [35] Cunha LC, Azeredo FS, Mendonça ACV, Vieira MS, Pucci LL, Valadares MC, et al. Acute and subacute toxicity studies of the latex and of the ethanolic extract of the leaves of Synadenium umbellatum Pax in rats. Rev Bras Farmacogn. 2009;19:403–11. Brought to you by | University of Fort Hare Library Authenticated | gotunola@ufh.ac.za author's copy Download Date | 5/28/18 8:46 AM 11