3.       Introduction 
An estimated 80 % of the world population depends on traditional medicine and plant-derived medicines for health care [1]. Intensive research on medicinal plants for bioactivity and lead compounds that could be developed into pure drugs for the treatment of ailments plaguing humans are been carried out in many laboratories worldwide [2]. The increasing consumption of natural products from plants as medicines and health supplements emphasizes the need for toxicity evaluation of medicinal plants. Efforts are being made in the area of herbal medicine to identify the bioactive compounds, elucidate their molecular structures, and establish their mechanisms of action and potential toxicological profile. 
Lophira lanceolata is a wild oil seed plant from savannah regions which grows up to 12 m tall with twisted short branches. The fruits and seeds are rich in oils mainly polyunsaturated fatted acids such as α-linoleic (>30 % w/w) and arachidonic (>14% w/w) acids [3] used as edible oils, in cosmetics, soap making and for medicinal purposes. Traditionally, its leaves are used to treat stomach pain and to control cough [4]. The leaves are natural aphrodisiac and fertility enhancer in males [5], and the infusion is used in treating malaria and jaundice [6]. The plant has been reported to have antidiabetic and antilipidemic [7], antiplasmodial and antioxidant [8], and anthelmintic. [9] activities. The leaves were found to contain flavonoids, anthraquinones, carbohydrate, glycoside, phenols, saponins, steroids, tannins, and free reducing sugar [10]. Considering the wide folkloric uses, as well as the reported biological activities of the leaf, this study was aimed at investigating sub-acute toxic effect of leaf extract of Lophira lanceolata employing some biochemical and hematological parameters. 
4.       Materials and Methods 
4.1.  Chemicals, Solvents and Reagents
All the chemicals and reagents used for this experiment were of analytical grade products of May and Baker (England) and Merck, Darmstadt (Germany).
4.2.  Animals 
Adult albino rats of either sex (100-150 g), obtained from the Animal House Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka were used for this study. Animal studies were conducted in compliance with the National Institute of Health Guide for Care and use of Laboratory Animals (Pub. No 85-23, revised 1985), and in accordance with the University of Nigeria Ethical Committee on the use of laboratory animals. The animals were housed in a well-ventilated room with a 12/12 h light/dark condition and ambient room temperature. They were maintained on standard feed pellets and water ad libitum throughout the duration of the experiment. 
4.3.  Equipment 
Heparinized and non-heparinsed capillary tubes, plastacine, test tubes, sample bottles, UV  Spectrophotometer (Merck, Germany), Incubator, Micro capillary tube (Marrenfeld,  Germany), Microheamatocit Centrifuge (Hawksley, England), Microheamatocit reader  (Hawksley, England), Heamocytometer Set, Diluting Pipette (Hawksley, England),  Automatic pipette (Superfit equipment Anies), Haemoglobinometer kit (Morienfeld,  Germany), Laboratory Tally Counter (Clay Adams, New Jersey), light Microscope (Leica  Inc, USA), Differential Cover Slips (Surgifriend Medicals, England).
4.4.  Collection 
Fresh leaves of Lophira lanceolata were collected in May 2013 from Nsukka, Nsukka Local Government Area of Enugu State, Nigeria. The plant was identified by Mr. Alfred Ozioko of International Center for Ethnomedicine and Drug Development (Inter CED) Nsukka. 
4.5.  Extraction 
The leaves of the plant were air-dried at room temperature and ground into powder using a grinder (ADDIS Nigeria). The powdered material (2.37 kg) was macerated with 4.5 liters of 70 % methanol for 72 h with constant shaking. The resultant mixture was filtered using Whatman No. 1 filter paper and the filtrate was concentrated to dryness in vacuum at 40^ºC using rotary evaporator. This gave a yield of 108.81g (4.59 % w/w). 
4.6.  Sub-acute Toxicity Study 
The animals were fasted overnight and were divided into four groups of five per group and treated as follows. Group 1 rats served as control and were given distilled water (10 ml/kg). Groups 2, 3 and 4 received 100, 400 and 1000 mg/kg of the extract administered orally for two weeks and the animals were regularly checked for any signs of toxicity. On day 0 and 14, blood was collected and subjected to hematological and biochemical tests. 
The total white blood cell counts, and differential white blood cell counts were carried out according to the method of Docie and Lewis [11] while the assays of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) activities were performed as described by Retaman and Frankel [12], while Alkaline Phosphatase (ALP) activity was determined by the phenolphthalein monophosphate method for in vitro determinations using Quimica Clinica Applicada (QCA) test kit, Spain [12]. 
4.7.  Body Weight, Food Intake, Water Consumption and Mortality 
Body weights were measured before the treatment, and on days 7 and 14 post treatment. Food and water intake were monitored. The animals were observed for possible physical and behavioural changes and mortality within the experimental period [13]. 
4.8.  Organ Weights 
The heart, liver and kidneys were carefully excised, examined macroscopically and weighed. 
4.9.  Statistical Analysis 
Results were expressed as mean ± SEM. Data obtained were analyzed by one-way ANOVA and subjected to Dunnet Post Hoc test using Graph Pad Prism Version 5. Differences between means were accepted significant at p < 0.05. 
5.       Results 
5.1.  Effect of L. lanceolata Leaf Extract on ALT, AST and ALP in Treated Groups of Rats 
Treatment with the methanol leaf extract of L. lanceolata did not cause any significant (p > 0.05) increase in serum alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase (Table 1).
5.2.  Effect of the Extract on Total White Blood Cell Count and Differential Leucocytes Counts 
Hematological analysis revealed no significant (p>0.05) change in the hematological parameters of the treatment groups as compared to the control group (Tables 2 and 3). 
5.3.  Effect of the Extract on Gross Morphology 
The animals were healthy with no differences being noted with respect to the control group. No significant changes were observed in the body weight of treated groups as compared to control and no mortality was observed during the experimental procedures. Oral administration of the extracts did not produce any symptoms of toxicity in rats. There were no deaths or any obvious signs of toxicity within the period. There was no evidence of changes in the skin, fur, eyes, sleep, salivation, faecal output and gross behaviour. Feed and water consumptions did not significantly alter, and the liver, kidney and heart weights were not significantly affected by treatment with the extract (Table 4). 
6.       Discussion 
Liver function tests are useful in the evaluation of hepatic dysfunction. Some of the biochemical markers usually considered are serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and ratio of aminotransferases, alkaline phosphatase, gamma glutamyl transferase, 5’ nucleotidase, ceruloplasmin and α-fetoprotein [14]. These enzymes and the end products of metabolic pathways are very sensitive and their elevated levels in the serum may serve as indication of liver damage. Predominantly raised alkaline phosphatase represents the cholestatic pattern of biliary pathology, while predominantly raised alanine and aspartate aminotransferases represent the hepatocellular pattern of hepatocellular pathology [15]. Beyond the liver function tests, prothrombin time provides another marker of liver synthetic function and a low platelet count suggests portal hypertension. 
In many developing countries, herbal medicines continue to receive attention as alternatives to synthetic Pharmaceutical products [16] and are generally considered safe and effective. In addition, bioactive compounds isolated from these herbal products are generally considered safe and used as over- the-counter products. Administration of these herbal medicines for a long period without expert delineating their potential side effects might be hazardous. [17].
In the present study, oral administration of the methanol leaf extract of L. lanceolata for 14 days did not elicit any signs of toxicity. Acute toxicity study of stem bark of L. lanceolata revealed oral LD₅₀ greater than 5, 000 mg/kg. [18] indicating the safety of this plant. 
Drug induced toxicity is one of the leading causes of hepatoxicity and the assessment of liver function serves as a diagnostic tool [19]. Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) are commonly assayed for in the serum to assess liver damage [20]. Unfortunately, extra hepatic injury such as muscle injury, can also lead to elevations in ALT, making ALT not entirely hepato-specific [21]. Despite the fact that extrahepatic injury can lead to increase in ALT, serum ALT remains the most widely used and universally accepted biomarker for liver damage, [22]. Oral administration of the methanol leaf extract of Lophira Lanceolata did not exert any significant change in the activities of AST, ALT and ALP enzymes suggesting no deleterious effect on the liver. 
The extract did not have any significant effect on the lymphocytes and total white blood cell counts, thus, the extract is devoid of any serious inflammation or damage to body cells, tissues and organs. 
Organ weight changes can be sensitive indicators of target organ toxicity, and significant changes in organ weights may occur in the absence of changes in other pathologic parameters [23]. In this study, the extract did not cause any significant change in the weight of the three vital organs, indicating the absence of target organ toxicity. From the previous studies 100 mg/kg of the extract has been found effective in malaria [8] and can be used for clinical studies and treatment. Moreover, since the LD₅₀ is above 5 g/kg, a dose up to 200 mg/kg is still acceptable. 
7.       Conclusion 
These results strongly suggest that the leaf extract of L. lanceolata is safe and well-tolerated and devoid of deleterious effects on the vital organs.

Table 1: Effect of the extract on ALT, AST and ALP.

Table 2: Effect of the extract on Total White Blood Cell Count.

Table 3: Effect of the extract on Differential Leucocytes Counts.

Table 4: Effect of the extract on gross morphology.

1.       Qazi MA, Molvi KI (2016) Herbal medicine: A comprehensive review. Int J Pharm Res 8: 1- 5.
2.       Chikezie PC, Ojiako OA (2015)  Herbal Medicine: Yesterday, Today and Tomorrow. Alt Integr Med 4: 195.
3.       Benjamin L, Amade O, Joseph IB, Anne ML (2017) Natural stands diversity and population structure of Lophira lanceolata Tiegh. ex Keay, a local oil tree species in Burkina Faso, West Africa. Handbook Flor Crop Sci 91: 85-96.
4.       Olotu PN, Olotu IA, Kambasha MB, Ahmed A, Ajima U, et al. (2017) Culture and traditional medicine practice among the Idoma people of Otukpo local government area of Benue State, Nigeria. Int Res J Pharm 8: 33-39.
5.       Mahima S, Dharmendra A, Kiran B, Gupta RS (2017) Natural aphrodisiac and fertility enhancement measure in males: A review. Cur Med Res Pract 7: 51-58.
6.       Aliou D, Honore S, Armand K, Gerard G (2017) Quantitative ethnobotany of Lophira lanceolata Teigh ex Keay (Ochnaceae) in Benin (West Africa). Int J Biol Chem Sci 11:1236-1253.
7.       Awede B, Houetchegnon P, Djego JG, Djrolo F, Gbenou J, et al. (2015) Effects of Lophira lanceolata and of three species of Gardenia leaves aqueous extracts on blood glucose and lipids in Wistar rat. J Phys Pharm Adv 5: 757-765.
8.       Onyeto CA, Akah PA, Nworu CS, Okoye TC, Okorie NA, et al (2014) Anti-plasmodial and antioxidant activities of         methanol extract of the fresh leaf of Lophira lanceolata (Ochnaceae). AJB 13: 1731-1738.
9.       Justin K, Dieudonne N, Djafsia B, Jacqueline DV, Eva L (2017) Phytochemical analysis and in vitro anthelmintic activity of Lophira lanceolata (Ochnaceae) on the bovine parasite Onchocerca ochengi and on drug resistant strains of the free-living nematode Caenorhabditis elegans. BMC Compl Alt Med 17: 1-12.
10.    Audu SA, Mohammed I, Kaita HA (2007) Phytochemical screening of the leaves of Lophira lanceolata (Ochnaceae). Life Sc J 4: 75-79.
11.    Dacie JV, Lewis SM (1975) Practical Haematology. 9th Edn. New York: Churchill Livingstone.
12.    Reitman S, Frankel SA (1957) Colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Path 28: 56-63.
13.    Mobolaji J, Muhammad HL, Makun AH, Busari BM, Abdullah SA (2016) Acute and subacute toxicity studies of aqueous and methanol extracts of Nelsonia campestris in rats. J Acute Dis 5: 62-70.
14.    Shivaraj G, Prakash BD, Vinayak VH, Avinash AKM, Sonal NV, et al. (2009) A review on laboratory liver function tests. Pan Afr Med J 3: 17.
15.    Christina L (2017) How to Interpret Liver Function Tests. Southern Sudan Med J 10: 40-43.
16.    Mythilpriya R, Shanthi P, Sachdanandam P (2007) Oral acute and subacute toxicity studies with Kalpaamruthaa, a modified indigenous preparation on rats.  J Health Sci 53: 351-358.
17.    Eran BA, Noah S, Lee HG, Kamer M, Suha O, et al. (2016) Potential risks associated with traditional herbal medicine use in cancer care: A study of Middle Eastern oncology health care professionals. Cancer 122: 598-610.
18.    Aba PE, Ozioko IE, Udem ND, Udem SC (2014) Some biochemical and haematological changes in rats pretreated with aqueous stem bark extract of Lophira lanceolata and intoxicated with paracetamol (acetaminophen). J Compl & Integr Med. 11: 273-277.
19.    Katzung BG (2003) Basic and Clinical Pharmacology 8th Edition Lange Medical Book Megram Hill, New York: 52.
20.    Dejong CH, Van de Poll MC, Soeters PB, Jalan R, Olde Damink SW (2007) Aromatic amino acid metabolism during liver failure. J Nutr 137: 1579S-1585S.
21.    Aguwa NC (2004) Liver Disease in: Therapeutic Basic of Clinical Pharmacy in the Tropics, 3rdEd. SNAPP Press Ltd, Enugu Nigeria 546-556.
22.    Solter P, Liu Z, Guzman R (2000) Decreased hepatic ALT synthesis is an outcome of sub chronic microcystin-LR toxicity. Toxicol Appl Pharmacol 164: 216-220.
23.    Bailey SA, Zideli RH, Perry RW (2004) Relationship between Organ Weight and Body/Brain Weight in the Rat: What is the Best Analytical Endpoint? Toxicol Path 32: 448-466.