JMBR VOLUME 15 Number 1 JUNE 2016

JMBR: A Peer-review Journal of Biomedical Sciences June 2016, Vol. 15 No. 1 pp 62-73

ACUTE AND SUBCHRONIC TOXICITY STUDIES OF ETHANOL EXTRACT OF TERMINALIA MACROPTERA STEM BARK IN WISTAR ALBINO RATS

Akpovona AE 1, Onoagbe IO1, Eze GI2 and Omonkhua AA 3

ABSTRACT This study evaluated the toxicity of ethanolic extract of Terminalia macroptera stem bark in female albino rats. Acute toxicity was determined by the Lorke Method while subchronic toxicity was determined by assessing some indicators of organ functions as well as lipid peroxidation levels after administering extract (50 -

1200 mg/kg) for 28 days. The extract exhibited LD50 > 5000 mg/kg. Subchronic investigation revealed slight cytolysis of hepatocytes but no significant effect on the hepatobiliary system. The synthetic role of the liver remained intact except in 400 - 1000 mg/kg dosed groups. Stable cardiological and nephrological states were indicated by heart and kidney function parameters respectively while malondialdehyde concentration was significantly elevated only in 200 mg/kg body weight dosed group when compared with control group. No distortions in liver tissue architecture were however observed. The study showed that extract from the stem bark of Terminalia macroptera is safe for consumption in rats but caution must be given to long-term repeated administration.

INTRODUCTION sufficient to eliminate the disease Intensive study of plant extracts has come conditions. A common plant used in the wake of the realisation that synthetic in tropical Africa for various chemical treatment of ailments may lead to treatments is Terminalia macroptera bioaccumulation of hazardous alien (commonly called “Orin idi odan” compounds. Plant phytochemicals are not among the Yorubas). It belongs to only therapeutic, but being organic are the Combretaceae plant family and readily absorbed in the body system. it is a member of the guinea savanna Traditional medicine practitioners who are biome1. Its leaves, flowers and stem engaged in the prescription of herbal have long been used in West Africa preparations do so with no empirical for the treatment of various diseases evidence of the side effects associated with such as tuberculosis and hepatitis 2-7. these medicinal plants or the safe dose just The extracts from the stem bark have been shown to depress food KEYWORDS: Terminalia macroptera, acute toxicity, 8 subchronic toxicity. intake and decrease body weight . 1Department of Biochemistry, Faculty of Life Sciences, Phytochemical evaluation of University of Benin, Benin City, Nigeria, 2Anatomy Department, School of Basic Medical Sciences, University of Terminalia macroptera stem bark Benin, Benin City, Nigeria and 3Medical Biochemistry Department, School of Basic Medical Sciences, University of extracts has revealed the presence Benin, Benin City, Nigeria. of bioactive components 9 - 1 1 . *Correspondence However, detailed scientific Ambrose E. Akpovona1 E-mail: [email protected], Tel: +2349081340988 studies of the plant's possible Iyere O. Onoagbe1 toxicity (within the available E-mail: [email protected], Tel: +2348037275378 literature) have not been carried Gerald I. Eze2 E-mail: [email protected], Tel: +2348033750269 out. This among other reasons have Akhere A. Omonkhua3 made a number of pharmaceutical E-mail: [email protected], Tel: +2348039286549

© CMS UNIBEN JMBR 2016:15(1) Acute and Subchronic Toxicity Studies of Ethanol Extract of Terminalia Macroptera Stem Bark in Wistar Albino Rats....63 industries in Africa which should be Medical Sciences, University of Benin, using this herb to be doubtful about its Benin City. The rats were maintained in potentials. There are arguments that raw well ventilated cages under standard parts of plants may be toxic due to the laboratory conditions of temperature possible release of hydrocyanic acids or 23°C (± 3°C), photo sequence of 12 hours wild metabolites occasioned by special light and 12 hours dark, and relative 12 metabolic pathways in the body . The humidity of 60 (± 5) % in the animal aim of this work therefore, was to house of the Department of Biochemistry, determine if the stem bark of Terminalia University of Benin. They were allowed macroptera plant is safe for consumption access to standard pelleted mash and when used for the various treatment of water ad libitum. The rats were allowed to ailments. acclimatize for one week prior to commencement of experiment. MATERIALS AND METHODS Acute Toxicity Study Collection of Plant Material The rats were randomized into 3 groups Matured stem barks of Terminalia c o m p r i s i n g 3 a n i m a l s e a c h . macroptera were obtained from open Predetermined test doses (10, 100 and forest in Ilorin and identified in the 1,000 mg/kg body weight) were Department of Plant Biotechnology, administered orally in a single dose to the University of Benin, Benin City (voucher rats by means of a gavage according to the number UBH 0232). T method of Lorke13. Thereafter, animals were observed for manifestation of Preparation of Plant Extract toxicity and mortality for 24 hours. In the The bark was washed, cut into bits, dried second phase, 3 rats were divided into 3 under shade to constant weight and groups of 1 rat each and were also treated pulverized. Powdery sample obtained with the extract at doses of 1,600, 2,900 was then filtered with a 2 mm screen. The crude extract was prepared by adding 800 and 5,000 mg/kg body weight orally. The median lethal dose (LD ) was then ml of absolute ethanol to 200 g of 50 pulverized Terminalia macroptera stem estimated by the geometric mean of the bark and exhaustively extracted (at a doses for which mortality ratios of 0/1 and temperature range of 8 - 15 OC in a 1/1 were obtained. refrigerator) for 72 hours by maceration and regular agitation of the sealed jar. The Subchronic Toxicity Study extract was evaporated to dryness in The animals were randomized into 6 vacuum at 40 oC under a pressure of 175 groups of 4 animals each representing the mbar with a rotary evaporator (Model: dose groups of 50 mg/kg, 200 mg/kg, 400 SM-52 CS-1, Surgifield Medical, England) mg/kg, 1000 mg/kg, 1200 mg/kg and under reduced pressure. control. The crude extract was orally administered daily for 28 days, while Experimental Animals control animals received distilled water in Adult female Wistar rats of albino strain of an equivalent volume of the maximum approximate weight range (170 - 200 g) test dose. The animals were also observed were obtained from a pure line breeder in for manifestation of toxicity and mortality Anatomy Department, School of Basic as in acute test.

Blood and Organ Collection Histopathological Study Upon termination of the experiment on The tissues (liver) were fixed with 10 % the 29th day, the animals were euthanized formal saline solution (3 - 5 days). They (by inhalation for 1-3 minutes) in a were later dehydrated by passing through chamber containing cotton boll wetted varying (increasing) concentrations of with drops of CHCl3. Blood was obtained alcohol, 70 % to 100 %, cleared in xylene by heart puncture by means of a 5 ml and then embedded in molten paraffin. hypodermic syringe and needle and Five micron (5 µm) microtome sections placed in 5 ml sterile sample container for were stained with hematoxylin and eosin biochemical assays. The blood was dyes. The sections were examined under allowed to stand at room temperature for light microscope at high power 30 - 45 minutes to clot and centrifugation magnifications and photomicrographs performed at 1000 x g for 5 minutes to taken28. obtain serum with an electric centrifuge- 12 buckets, model 80 - 2 (Finlab Nigeria Statistical Analysis Ltd). The liver tissues were extracted, Results were analyzed statistically by one- rinsed in physiological saline and dried way ANOVA followed by Duncan's between layers of Whatman filter paper. multiple range test by using SPSS version 19 (2010). All values were expressed as Biochemical Analyses the mean ± SEM. P < 0.05 compared to The separated serum was analyzed for control was considered to be statistically various biochemical parameters: alanine significant. aminotransferase and aspartate aminotransferase activities14, total protein RESULTS concentration 1 5 , serum albumin There was no record of mortality for the concentration16 while the amount of acute toxicity investigation. However, globulin was calculated as a difference somnolence was observed in the groups between total serum protein and serum administered 1000 - 5000 mg/kg body albumin. Other biochemical parameters weight, which reverted to normal agility include serum bilirubin concentration17, after 4 hours. Mean lethal dose of the y-glutamyl transferase activity16, alkaline extract was found to be above 5000 mg/kg phosphatase activity18, lactate dehydrogenase body weight. activity19, creatine kinase activity20, creatinine concentration21, serum urea concentration22, Table 1 shows the effect of the extract on sodium23-24, chloride25, potassium26 and aspartate aminotransferase and alanine evaluation of the liver homogenate for aminotransferase activities, total protein, membrane lipid peroxidation27. albumin, globulin, direct and total bilirubin concentrations as well as y- Preparation of Tissue Homogenates glutamyltransferase and alkaline Tissues of liver were homogenized phosphatase activities of the rat liver respectively in ice cold normal saline enzymes present in serum after 28 days of (1:10 w/v) and centrifuged at 10,000 rpm administration. Extract doses of 200 for 15 minutes. The supernatant was mg/kg and 1000 mg/kg caused significant stored at 4 oC and analysized within 24 (p < 0.05) increase in AST activities when h o u r s f o r m a l o n d i a l d e h y d e compared with control group. Extract concentration. concentrations of 200 mg/kg, 400 mg/kg,

1000 mg/kg and 1200 mg/kg caused induced reduction in the concentration of significant (p < 0.05) increases in ALT serum total protein, where only those of activities that were not dose dependent 400 mg/kg and 1000 mg/kg were when compared with control group. significantly reduced (p < 0.05), an observation which was similar for There was significant (p < 0.05) elevation albumin and globulin. The administered in the concentration of total protein in the dose of 200 mg/kg concentration of extract serum of rats dosed with 1200 mg/kg body caused a significant (p < 0.05) depression weight extract of ethanol when compared in blood albumin with a contrasting with control. However, all other doses significant (p < 0.05) elevation in

© CMS UNIBEN JMBR 2016:15(1) Acute and Subchronic Toxicity Studies of Ethanol Extract of Terminalia Macroptera Stem Bark in Wistar Albino Rats....69 globulin content. This trend was also (p < 0.05) were reflected in the urea and repeated in the group dosed with 1200 sodium ion concentrations of all test mg/kg body weight. No significant groups administered the extract. differences were observed in total bilirubin concentrations except in the Administered doses of 400 mg/kg to 1,200 group dosed with 1200 mg/kg which mg/kg showed contrasting changes in the showed significant (p < 0.05) reduction alteration of concentrations between with respect to control group. sodium and chloride ions. The extract doses (200 mg/kg and 400 mg/kg) caused The activities of y-glutamyl transferase significant reduction (p < 0.05) in the concentration of potassium ion, but no were significantly decreased when significant (p < 0.05) differences were compared to control group while those of noted in the groups administered 50, 1000 alkaline phosphatase were significantly and 1200 mg/kg of the extract when increased (p< 0.05) in groups compared with control group. administered lower doses. Their activities were not altogether altered in the highest The concentrations of malondialdehyde dosed group. in rats administered ethanolic extracts are shown in figure 3. Test animals Figure 1 shows the lactate dehydrogenase administered 200 mg/kg extract showed activities in serum of control and tested significant (p < 0.05) elevated levels in rats. The activity of the enzyme showed concentration of MDA when compared to no variance with that of the control, control group respectively. except for the enzyme activity in the group administerd 400 mg/kg which Figure 4 shows photomicrographic plates reflected significant (p < 0.05) reduction. A to H of liver sections of control and test Figure 2 shows creatine kinase (CK) rats administered extracts of Terminalia activities in rats administered ethanolic macroptera stem bark. extract of Terminalia macroptera stem DISCUSSION bark for 28 days. Doses 200 mg/kg, 400 One of the toxicological indices used to mg/kg, 1000 mg/kg and 1200 mg/kg assess the safety of plant extracts is Lethal ethanolic extract administered groups Dose 50 % (LD50), which is the amount of experienced significant reduction (p < acute dose of the drug required to kill half of 0.05) in CK activities when compared the test population. In this study, 5000 mg/kg with control group respectively. body weight of the extract did not reflect any observable toxic symptoms under 24 hour Table 2 shows the kidney function tests of monitoring. The result of this study clearly r a t s a f t e r 2 8 d a y s o f e x t r a c t corroborated previous work that aqueous administration. Creatinine concentration extract of Terminalia macroptera stem bark are relatively safe at 2000 mg/kg10. This of the test groups administered ethanolic present study showed no record of mortality extract were not significantly altered (p < with ethanolic extract, however, the group of 0.05) when compared with control group rats dosed with 1000 mg/kg to 5000 mg/kg except for 50 mg/kg dosed group that was body weight crude extract showed significantly higher (p < 0.05) than somnolence (animals appeared drowsy, but control. However, significant elevations on prodding were aroused, moved few

© CMS UNIBEN JMBR 2016:15(1) 70....Journal of Medicine and Biomedical Research sluggish steps and resumed sleeping determination is considered more positions) for 1 to 4 hours after sensitive when compared to alkaline administration. This is in consonance with phosphatase since it is primarily from the the findings of Adebiyi and Abatan29, who hepatobiliary system31. The results reported that 1000 mg/kg - 3000 mg/kg showed reduced activities observed in the ethanolic extracts of Enanthia chlorantha y - glutamyltransferase (GGT) enzyme stem bark caused reduced mobility, dullness assay which did not complement the and general weakness in albino rats within increase in alkaline phosphatase (ALP) 24 hours of administration. activities seen only in rats administered 50 mg/kg (significant) and 200 mg/kg Although alanine aminotransferase (ALT) extracts (insignificant). The lack of activities were significantly elevated just consistency in the enzyme activities as aspartate aminotransferase (AST) across the doses may be due to intrinsic activities were staggeredly elevated in a factors in vivo which clearly did not point few dosed groups (200, 1000 and 1200 to hepatobiliary damage, but rather a mg/kg b. wt), ALT to AST ratios (not probable bone effect, an argument that shown in table) remained within non- can only be substantiated by bone pathological range indicating that histomicrography (not considered in this increased ALT levels may just be a slight study). The liver parameters showed insult on the integrity of the liver or a much isolated increase and decrease in physiological cause at its best. This dosed animals that reflected a case of observation was further buttressed by the nonmonotonic dose response 3 2 - 3 3 , significant decrease in the concentrations however, the group administered the of bilirubin and the non-alteration in the maximum dose showed no evidence of concentration of albumin, an indication hepatotoxicity from the assay results. of a stable synthetic function of the liver The activities of lactate dehydrogenase in the presence of the extract at its highest and creatine kinase were completely administered dose. In this present study, stable in the presence of ethanol extract of 1000 mg/kg body weight of extract had Terminalia macroptera, an indication of shown clear cut indication of no toxic effect on the heart muscle. hepatotoxicity by the significant increases in aspartate aminotransferase, The insignificant increase (p < 0.05) alanine aminotransferase, total bilirubin observed in the creatinine concentration and significant decreases in total protein, did not confirm the suspicion that the albumin and globulin, but definitely no ethanolic extract is toxic to the hepatobiliary damage as reflected by y- ultrafiltration apparatus of the kidney as glutamyltransferase (GGT) and alkaline phosphatase (ALP) activities. This posed by the significantly elevated serum observation seems to give support to our urea concentration of the test groups. previous study where 1000 mg/kg body However, the creatinine result may weight of extract caused the most of foretell of possible deleterious effect in depression in body weight of rats8. the kidney upon prolonged administration. The observation with urea concentration GGT is of clinical value in identifying the may be due to decreased blood flow to the source of obscure ALP elevations whose kidneys arising from stress in handling activity is usually high in hepatobiliary the animals. This appeared to be d i s o r d e r a n d b o n e d i s e a s e s 3 0 . supported by the hypernatraemia Consequently, y-glutamyltransferase observed in the extract dosed groups.

However, an onset of renal dysfunction for the generality of inconsistency resulting from the inability of the kidney observed in the nonmonotonic dose to regulate electrolyte balance may not be response of the investigated extract32. totally denied since low chloride and potassium concentrations were recorded Malondialdehyde (MDA) is a secondary for the test groups, except for the non- product of lipid peroxidation, that causes significant changes reflected by the cross-linkage of membrane components highest dose group which summarily containing amino groups which makes portrayed a current stable kidney the membrane fragile and easily function. perforated 3 6 . The monitoring of malondialdehyde levels in biological Physiologically, the circulatory system of system can be used as an important test animals provides a unique transport indicator of lipid peroxidation in vitro route for the plant phytochemicals and in vivo for various diseases. The mild making them come in contact with the elevation in concentration of liver MDA organs that are most susceptible to their observed in a few dosed groups of rats chemical influence. On arrival at a choice may be due to peroxidation activity organ, a dorminant component or group arising from injury to hepatic cells o f c o m p o n e n t s p r e s e n t i n t h e occasioned by repeated daily dosing. administered bolus, may have enjoyed an C o n s e q u e n t l y s u p p o r t i n g t h e advantage in terms of large quantity or an aforementioned, liver biomarkers effective bioactive structure when (alanine aminotransferase) might compared to other phytocomponents and probably have leaked through membranes consequently exhibited a progressive or of hepatocytes. regressive trend in observed effect under However, changes were not observed in the dose response experiment. Sudden the architectural layout of the liver tissue deviation in the trends observed upon probably as a result of the minuteness of the administration of the graded doses the membrane puncture. The presence of could be explained by the presence of an mild inflammatory cells detected in the active component (initially minor and liver slides auguments this argument. inactive at a lower dose), that just attained E q u a l l y, t h e c o n g e s t i o n a n d its effective bioactive level (EBL) at a lymphocytosis so observed in the higher dose in vivo irrespective of its ratio photomicrographs indicated a diffused to the domineering component at the activation of the local immune system of 34 former dose(s) . At this point, 'the the liver, which is a desirable effect of the nouveau active' either strengthened the extract, though. These observations observed dose response trend initiated at however, send precautionary signals on the lower doses or altered it. The effective the repeated administration of ethanolic bioactive component at the new dose extract of Terminalia macroptera stem became the dorminant component or at bark. its best competed with the initial dose frontliner and where possible shared their CONCLUSION dorminance at the receptors in the The study has shown that the LD50 of manipulation of the target organ. This ethanolic extract of Terminalia most likely explains the multimodal effect macroptera stem bark is greater than 5000 35 of plant extracts and may be responsible mg/kg body weight of Wistar albino rat

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