Accepted Manuscript Title: Toxicity assessment of Erythrophleum ivorense and Parquetina nigrescens Author: Louis Adu-Amoah Christian Agyare Emelia Kisseih Patrick George Ayande Kwesi Boadu Mensah PII: DOI: Reference: S2214-7500(14)00043-2 http://dx.doi.org/doi:10.1016/j.toxrep.2014.06.009 TOXREP 42 To appear in: Received date: Revised date: Accepted date: 10-5-2014 23-6-2014 23-6-2014 Please cite this article as: L. Adu-Amoah, C. Agyare, E. Kisseih, P.G. Ayande, K.B. Mensah, Toxicity assessment of Erythrophleum ivorense and Parquetina nigrescens, Toxicol. Rep. (2014), http://dx.doi.org/10.1016/j.toxrep.2014.06.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Toxicity assessment of Erythrophleum ivorense and Parquetina nigrescens Louis Adu-Amoah1, Christian Agyare1*, Emelia Kisseih2, Patrick George Ayande3, Kwesi Boadu Mensah4, 1 Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 2 us cr ip t Institute for Pharmaceutical Biology and Phytochemistry, University of Muenster, Corrensstrasse 48, D-48149, Muenster, Germany 3 Department of Human Biology and Nursing, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana 4 Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana M an *Corresponding author: Dr. C. Agyare, Department of Pharmaceutics, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. Telephone: +233246369803. E-mail: cagyare.pharm@knust.edu.gh Abstract d Erythrophleum ivorense and Parquetina nigrescens are found growing in tropical regions and te they are used in African traditional medicine to treat various ailments including wounds, boils and anaemic conditions. Some species of plant in the Erythrophleum genus are also known to ep be poisonous and toxic to several livestock. However, there is no information on the toxicity of E. ivorense and P. nigrescens. This study is to determine the cytotoxicity and subchronic toxicity properties of methanol leaf extract (EIML) and methanol stem barks extract (EIMB) Ac c of E. ivorense and methanol leaf and aerial part extract of P. nigrescens (PNML). Concentrations from 0.1 to 100 µg/mL of the extracts were used to determine the influence of the extracts on the release of lactate dehydrogenase (LDH) from HaCaT keratinocytes. The EIML and EIMB extracts showed increase in LDH released from HaCaT keratinocytes at 0.1 to 10 µg/mL and 1 to 100 µg/mL for the PNML extracts (p>0.05). Wistar rats were orally administered with 100, 300 and 1000 mg/kg body weight of the extracts (EIML, EIMB and PNML) for 35 days. Tissues from the kidney and liver of the rats treated with lower doses (100 to 300 mg/kg body weight) of EIML extract showed highly vascularized kidneys with numerous glomerular tufts, healthy hepatocytes and sinusoids in liver. However, there were persistent renal tissue inflammation and glomerular degeneration in kidney, and increased 1 Page 1 of 23 inflammatory infiltrates with few vacuolations and scarrings in liver in rats treated with higher extract dose of 1000 mg/kg body weight of rat. The rats treated with EIMB extract showed persistent renal and hepatocyte inflammations with glomerular and hepatocyte necrosis at all administered doses (100, 300 and 1000 mg/kg body weight) which are indications of renal and hepatic toxicities. Though rats administered with 100 and 300 mg/kg of PNML extract showed renal haemorrhage and inflammation and hepatic inflammation, the us cr ip t rats administered with 1000 mg/kg body weight showed restoring glomerular tufts and improved vasculature and liver with reduced inflammatory infiltrates with healthy hepatocytes. Phytochemical screening of EIML, EIMB and PNML extracts revealed the presence of alkaloids, tannins, flavonoids, sterols, cardiac glycosides and terpenoids. an Key words: Wistar rats, toxicity, lactate dehydrogenase, cytotoxicity, HaCaT keratinocytes M 1.0 Introduction Traditional medicine practice is a prominent aspect of the primary healthcare systems in most d developing countries. It is estimated that about 80% of the world’s population especially te people from developing countries depend on traditional medicines of plant origin (Nath et al., 2011). The inclusion of plants in traditional medicines dates back to several thousands of ep years (Abu-Rabia, 2005) and it is on the surge due to their great source of bioactive compounds employed in pharmaceutical intermediates and chemical agents for synthetic drugs (Nath et al., 2011), and the fact that they are cost-effective and lack the complexity of Ac c compounded pharmaceutical preparations (Wolf, 1999; Zirihi et al., 2005). However, many herbal preparations and traditional folk medicines have not been thoroughly tested or investigated (Kunwar et al., 2009). Erythrophleum ivorense (A Chev.) is a large tree found growing in tropical regions in Africa including Ghana, Cote d’Ivoire and Liberia. It is also described as the ‘ugly’ plant. It can grow up to 40 m tall, usually bole cylindrical, but it may occasionally be fluted at the base, with or without buttresses at old age. The diameter is usually 60 to 90 cm (Irvine, 1961; Burkill, 1995). Parquetina nigrescens (Afzel.) Bullock belongs to the family Ascelpiadaceae. It is a slender, glaborous twining shrub that grows up to tops of forest trees. The plant is present in low 2 Page 2 of 23 bushes in savannah areas, farm clearings in forests and transition forests in West African countries including Ghana and Cote d’Ivoire (Irvine, 1961). The leaves and roots of P. nigrescens are used as poultice to treat wounds, boils, carbuncles and worm infections in ethno-medicine (Agyare et al., 2009). Aside from the therapeutic values of some of the compounds from plants, others are also known to be toxic and harmful to humans and animals. Several Erythrophleum spp. including us cr ip t E. lascianthum and E. guineense have been studied to be toxic to livestock and humans (Adeoye and Oyedapo, 2004). They have been used as hunting poisons for animals and suffering or test drinks for people convicted of serious crimes in some communities (Dongmo et al., 2001). The kidney and liver are organs of metabolism and excretion, respectively, of xenobiotic molecules such as saponins, alkaloids, tannins etc. (Irvine, 1961). These organs can suffer an from diverse diseases or disorders (Wolf, 1999). Lactate dehydrogenase enzymes are cytoplasmic in origin in intact cells of animals. However, these enzymes may leak through M phospholipid membrane channels of cytoplasm of cells and be measured extracellularly, indicative of injury or damage to the cells. Plants possess several metabolites with varied pharmacological effects. The need to evaluate the potential toxicity of these compounds and d that of many therapeutic molecules has led to the development of various cytotoxicity assays te (Todd et al., 1999). The study was therefore to investigate the cytotoxicity of methanol leaf and aerial parts extract of P. nigrescens and methanol leaf and stem bark extracts of E. ep ivorense on HaCaT keratinocytes and in vivo toxicity effect the extracts on kidney and liver Ac c tissues of Wistar rats. 2.0 Materials and methods 2.1 Plant materials and chemicals The leaves and barks of E. ivorense were collected from the Botanic Garden, University of Ghana, in November 2011 by Mr. John Yaw Amponsah. The leaves and aerial parts of P. nigrescens were collected by Mr. Eric Gyebi from Jachie in the Bosomtwi District of the Ashanti Region, Ghana, in December 2011. The plant materials were authenticated by Dr. Alex Asase, Department of Botany of University of Ghana, Legon, Ghana. Voucher 3 Page 3 of 23 specimens of the plant materials have been kept in the Ghana Herbarium, University of Ghana, Legon, Accra, Ghana. Unless stated otherwise, all the chemicals were purchased from GPR, BDH, Poole, UK. 2.2 Preparation of extracts Fresh leaves and stem bark of E. ivorense and leaves and other aerial parts of P. nigrescens us cr ip t were washed with tap water to remove debris and soil particles. The plant materials were dried at room temperature (28-30ºC) for 6 days before the dried plant materials were powdered using laboratory mill machine (Type 8, Christy and Norris Limited, UK). Eight hundred grams (800 g) of the powdered leaf, 650 g of powdered bark of E. ivorense and 700 g of powdered leaf and other aerial parts of P. nigrescens were and each soaked in 2.5 L of 70% v/v methanol in a stoppered container. These were shaken for about 5 min and an left to extract by means of maceration (shaking the mixture intermittently) at 28ºC for 72 h (Parekh et al., 2006). The mixtures were filtered into a porcelain crucible using a fine mesh. The supernatant were concentrated below 40ºC using rotary evaporator and then lyophilized. M The yields of the methanol leaf extract of E. ivorense (EIML), methanol bark extract of E. ivorense (EIMB), and methanol leaf and other aerial parts extracts of P. nigrescens (PNML) 2.3 Phytochemical analysis te d were determined. ep The phytochemical constituents of the methanol extracts were determined using methods described by Trease and Evans (2009) and Harborne (1988). Ac c 2.4 HPLC profile of methanol extracts Modified methods of Srivastava et al. (2004) and Ding et al. (2011) was used to determine the HPLC profile of the methanol extracts (EIML, EIMB and PNML) using a reverse phase Jupiter C18 300R column (250 x 4.6 mm). Concentrations of 10 mg/mL of extracts were prepared with methanol-water (3:7 v/v) which is the same as the mobile phase and a volume of 10 µL injected into the columns. The run time for the column of extracts was 10 min at 22°C under a pump pressure of 21MPa and flow rate of 1.0 mL/min. The resultant chromatograms were observed at a wavelength of 254 nm. The retention times and area under curve of the chromatograms were then determined. 4 Page 4 of 23 2.5 Ethical approval for animal studies Ethical clearance and approval for the subchronic toxicity studies in Wistar rats was given by the Ethical Committee on Animals of the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana in accordance with the Guide for Care and Use of Laboratory Animals, NIH, Department of Health Services Publication, USA, no. 83-23, revised 1985 (Garber et al., us cr ip t 2010). Approval of cytotoxicity studies was made by the local Ethical Committee of University of Muenster, Muenster, Germany (2006-177-f-S). 2.6 Handling and preparation of test laboratory animals Fifty (50) male Wistar rats of weight ranging between 95 to 150 g were obtained from the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame an Nkrumah University of Science and Technology, Kumasi, Ghana and housed in stainless steel cages containing wood shavings for bedding. They were to acclimated holding facilities M for 2 weeks before experimental procedure was started. The rats were randomly divided into 10 groups (5 rats per group), which consisted of 9 treatment groups and one untreated group (control). They were fed on normal commercial dietary pellets (GAFCO, Tema, Ghana) and d given tap water ad libitum during the study. Environmental conditions were maintained at a te temperature of 29±2°C and a relative humidity of 40±10% with 12 h light/dark cycle. ep 2.7 Administration of extracts to animals The rats were fasted for 12 h prior to administration of doses. The extracts were administered Ac c to the rats as aqueous suspensions of finely grounded powder for thirty-five (35) days via oral gavage, using a curved, ball-tipped stainless steel feeding needle connected to a syringe at the concentrations. Each group was made of five rats. Group 1 rats were treated with 100 mg/kg of methanol leaf extract of Erythrophleum ivorensis (EIML); Group 2 rats were treated with 300 mg/kg of EIML; Group 3 rats were treated with 1000 mg/kg of EIML; Group 4 rats were treated with 100 mg/kg of EIMB; Group 5 rats were treated with 300 mg/kg of EIMB; Group 6 rats were treated with 1000 mg/kg of EIMB; Group 7 rats were treated with 100 mg/kg of methanol leaf and other aerial part extract of Parquetina nigrescens (PNML); Group 8 rats were treated with 300 mg/kg of PNML; Group 9 rats were treated with 1000mg/kg of PNML and Group 10 rats were administered with distilled water. 5 Page 5 of 23 2.8 Biochemical tests for determination of toxicity The animals were fasted overnight prior to necropsy and blood collection. The animals were anaethesized prior to euthanization and then decapitated after neck dislocation. Blood samples were taken through the jugular veins in the animals in each group into complete blood count (CBC) bottles containing ethylenediaminetetraacetic acid (EDTA-2K). t Haematological analyses which measured parameters such as red blood cell count, us cr ip haemoglobin concentration, hematocrit, mean corpuscular cell volume, mean corpuscular cell haemoglobin, mean corpuscular cell haemogolobin concentration, platelet count, white blood cell count, and differential WBC count were determined using automatic haematology analyser (Hitachi 7060, Japan). Portions of uncoagulated blood were centrifuged at 3000 rpm for 10 min and analyzed using a 7060 autoanalyzer (Hitachi, Tokyo). Serum biochemical indicators such as glucose, total an cholesterol, blood urea nitrogen (BUN), creatinine, total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total M bilirubin, creatine kinase, albumin/globulin ratio, triglycerides, phosphorus, calcium and 2.9 Histopathological studies d chloride were measured. te Histopathological examinations were performed on the kidney and liver of both treated and untreated Wistar rats as described by methods by Wasfi et al., 1994 and Guntupalli et al., ep 2006. The tissues were fixed in 10% formalin. They were then dehydrated sequentially in ethanol concentrations of 50 to 100%. The tissues are then rinsed in xylene to remove the Ac c dehydrant (ethanol) and finally embedded in paraffin for strengthening and easy dissection. Proir to sectioning the tissues, they are ‘de-paraffinized’ by rinsing in xylene, followed by washing in decreasing concentration of ethanol (100% to 50%) before rehydrating the tissues with water. Tissue sections of thickness 6 µm were made and stained with hematoxylin-eosin (H-E) dye to impart contrast for photomicroscopic viewing. The tissues were then observed under a light microscope at magnification (x600). 2.10 In vitro cytotoxicity Cytoplasmic enzyme lactate dehydrogenase (LDH) released from the cytosol of HaCaT keratinocytes when damaged or under stress was determined according to the method 6 Page 6 of 23 described by Agyare et al. (2011). Concentrations (0.10, 1.0, 10.0, 50.0 and 100.0 µg/mL) of the extracts in 100 µL HaCaT keratinocytes medium were made in the wells of 96-well microtitre plates and each well seeded with 105 keratinocyte cells and then incubated for 48 h at 35ºC with 5% of CO2. After incubation 25 µL of this supernatant was pipetted into 96-well microtitre plate and 25 µL lysis buffer added to both the supernatant and the adherent lysed cells in the wells. This was incubated at 28°C for 1 h and frequently agitated. Afterwards, 25 us cr ip t µL of substrate mix was added to both the untreated supernatant and lysed cell medium in the microtitre plate and incubated in dark at 20ºC for 30 min. The reactions were finally halted by the addition of 10 µL HCL-isopropanol solution to each well. The above procedure was repeated for untreated HaCaT keratinocytes and 10% Triton X-100 in FCS as negative and positive controls respectively. After incubation for 4 h measurement of LDH enzyme released an into the 25 µL supernatant and the absorbance were determined at 450 nm against 690 nm. 2.11 Statistical analysis Haematological and serum biochemistry data were expressed as Means ± standard error using M Graph Pad prism version 5.0 windows (Graph Pad Software, San Diego, CA, USA). A oneway analysis of variance (ANOVA) was done for the data followed by Newman-Keuls post- te d test. The values of p˂0.05 were considered to be statistically significant. 3.0 Results ep 3.1 Phytochemical screening The preliminary phytochemical screening of the methanol extracts (EIML, EIMB and Ac c PNML) revealed the presence of secondary metabolites including saponins, tannins, flavonoids and alkaloids (Table 1). The yields of EIML, EIMB and PNML extracts were 9.76, 15.54, 7.68 % w/w related to the dried plant materials, respectively and the yield was determined by dividing the final powdered extract (in grams) by the total weight of the dried plant material (in grams) multiplied by 100%. 7 Page 7 of 23 Table 1: Phytochemical constituents of methanol extracts of E. ivorense and P. nigrescens. EIML EIMB PNML Saponins + + + Hydrolysable tannins - + - Condensed tannins + - + Alkaloids + + + Terpenoids - + Sterols + - + Flavonoids + + + Cardiac glycosides - + + t Phytochemical us cr ip + an Key: (+) = presence of secondary metabolite; (-) = absence of secondary metabolites 3.2 HPLC profile M The HPLC profile of the methanol extracts were determined using different solvent systems. These HPLC profiles are used as identification for the plants. Different peaks in the chromatograms represent different compounds or constituents present in the plant extract ep Retention Time 12.319 4.295 3.704 2.893 3.002 7.258 2.064 2.202 10 5 1.153 5 1.645 Ac c 0.694 0.955 10 15 mAU UV1000-254nm 15 mAU te d (Fig. 1 to 3). 0 0 2 0 4 6 8 10 12 14 Minutes Fig. 1: HPLC profile of methanol leaf extract of E. ivorense at λ 254 nm. 8 Page 8 of 23 20 20 UV1000-254nm 4.530 t 0 2 4 6 8 Minutes us cr ip 0 mAU 10 3.950 1.982 2.068 2.215 2.428 2.762 1.268 1.482 0.695 10 1.088 mAU Retention Time 10 12 0 14 Fig. 2: HPLC profile of methanol bark extract of E. ivorense at λ 254 nm. UV1000-254nm 2 4 mAU 40 20 8.188 M 6 te 0 d 0 5.158 4.148 4.308 1.529 1.611 1.648 1.382 20 2.066 2.272 2.495 2.822 3.202 3.455 mAU 7.382 40 an Retention Time 0 8 10 12 14 Minutes ep Fig. 3: HPLC profile of methanol leaf and aerial parts extract of P. nigrescens at λ 254 nm Ac c 3.3 Subchronic toxicity studies Sections of liver and kidney tissues from rats of all 10 groups were made and histopathological studies done on them. The tissues of animals from treated group showed diverse morphological changes as compared to tissues sectioned from untreated group indicative of toxicity effects of the extracts administered. The renal tissues showed inflammation glomerular degeneration, hyalinized tissues and haemorrhage (Fig. 4). The hepatic tissues revealed scarring, inflammation, occlusion or congestive veins and necrosis (Fig. 5). 9 Page 9 of 23 t us cr ip an M d te Fig. 4: Effects of E. ivorense leaf and stem bark and P. nigrescens extracts on kidney tissue of treated and untreated Wistar rats. (G1) EIML 100mg/kg: Improved vasculature and a good Ac c ep number of glomerular tufts with no notable changes. (G2) EIML 300 mg/kg: Highly vascularized kidney with a good number of glomerular tufts. (G3) EIML 1000 mg/kg: Persistent renal tissue inflammation and glomerular degeneration, evident of reduced immunity. (G4) EIMB 100 mg/kg: Diseased kidney with profuse renal tissue inflammation, glomerular necrosis and vacuolation, evident of kidney degeneration. (G5) EIMB 300 mg/kg: Profuse glomerular degeneration with persistent renal tissue inflammation, indicative of persistent poor treatment response. (G6) EIMB 1000 mg/kg: Persistent renal toxicity with profuse haemorrhage, glomerular degeneration, reflective of treatment failure. (G7) PNML 100 mg/kg: Persistent hyalination and haemorrhage with focal inflammatory cells infiltration indicative of persistent kidney damage due to reduced immunity. (G8) PNML 300 mg/kg: Profuse haemorrhage and persistent renal tissue inflammation reflective of poor immune response. (G9) PNML 1000 mg/kg: A good number of restoring glomerular tufts with improved vasculature suggestive of recovery. (G10) Control 000 mg/kg: Normal kidney highly vascularized with a good number of glomerular tufts. Legend: RnT: Renal Tissue; RV: Reduced Vacuolation; TV: Tissue Vasculature.AS: Apoptotic Space; DG: Degenerating Glomeruli; G: Glomeruli; GT: Glomerular Tubules; H: Haemorrhage; HT: Hyalinized Tissue; IC: Infiltrating Cells; IT: Inflamed Tissue NG: Necrotic Glomerulus; OT: Occluding Tubules. 10 Page 10 of 23 t us cr ip an M d te ep Fig. 5: Effects of E. ivorense leaf and stem bark and P. nigrescens extracts on liver tissue of both treated and untreated Wistar rats. (G1) EIML 100 mg/kg: Healthy hepatocytes and Ac c sinusoids, indicative of effective treatment response. (G2) EIML 300 mg/kg: Marginal inflammatory infiltrates with healthy hepatocytes and sinusoids, indicative of recovery. (G3) EIML 1000 mg/kg: Increased inflammatory infiltrates with few vacuolations and scarrings, indicative of persistent disease. (G4) EIMB 100 mg/kg: Diseased liver with profuse inflammatory cell infiltration and hepatocyte necrosis. (G5) EIMB 300 mg/kg: Inflamed liver with hepatocyte necrosis, suggestive of sustained hepatotoxicity. (G6) EIMB 1000 mg/kg: Sustained inflammation with satellite hepatocyte necrosis, suggestive of impending hepatotoxicity. (G7) PNML 100 mg/kg: Increased inflammatory infiltrates with scarring and central venous congestion suggestive of poor immunity. (G8) PNML 300 mg/kg: Persistent inflammation with few scarrings and occluding vasculature, evident of sustained disease. (G9) PNML 1000 mg/kg: Reduced inflammatory infiltrates with healthy hepatocytes and tissue proliferation, indicative of liver restitution. (G10) Control 000 mg/kg: Normal liver with healthy hepatocytes and sinusoids. Legend: AS: Apoptotic Space; CV: Congested Vein; HN: Hepatocyte Necrosis; HP: Hepatocytes; IC: Inflammatory Cells; IH: Inflamed Hepatocytes; RT: Regenerating Tissue; S: Sinusoids; SN: Satellite Necrosis; ST: Scar Tissue; V: Vacuolation. 11 Page 11 of 23 3.4 Cytotoxicity studies Concentrations of 0.1, 1 and 10 µg/mL of the EIML and EIMB extracts and 0.1 to 100 µg/mL of the PNML extracts showed increases in the amount of LDH released from the HaCaT an us cr ip t keratinocytes as compared to the untreated cells. Ac c ep te d M Fig. 6: Influence of methanol leaf extract of E. ivorense on the release of LDH from HaCaT keratinocyte cells. UC = untreated cells Fig. 7: Influence of methanol bark extract of E. ivorense on the release of LDH from HaCaT keratinocyte cells. UC = untreated cells 12 Page 12 of 23 1 0.8 0.6 0.4 t 0.2 us cr ip % LDH released relative to the untreated control 1.2 0 Concentration (µg/ mL) an Fig. 8: Influence of methanol leaf and other aerial part extract of P. nigrescens on the release of LDH from HaCaT keratinocyte cells. UC = untreated cells M 3.5 Subchronic toxicity studies Several haematological and serum biochemistry parameters were measured for possible d indication or otherwise of end organ subchronic toxicity in the Wistar rats. The mean white blood cell counts measured in rats treated with the extracts were lower than the untreated rats te but red blood cells, haemoglobin and haematocrits concentrations of the animals in the treated groups were higher than those of the untreated groups (Table 2). In the serum ep biochemistry analysis, animals treated with the methanol extracts (EIML, EIMB and PNML) showed higher concentrations of albumin, cholestrol and total bilirubin (Table 3). The values Ac c obtained for the negative controls or from the untreated animals with respect to the serum and haematological parameters investigated were consistent with reference values for rats from literature values (Lang, 1993; Meingassner and Schmook, 1990). 13 Page 13 of 23 us cr ip t Table 2: Effects of methanol extracts on haematological parameters of Wistar rats in sub-chronic toxicity studies. Extracts and their concentration (mg/kg body weight) Parameters EIML 300 EIML 1000 EIMB 100 EIMB 300 EIMB 1000 PNML 100 WBC X 103/µL 11.0±0.50 11.2±0.60 7.18±0.75 16.2±0.12 13.2±1.00 12.1±0.74 8.20±0.47 * 8.16±1.11 * 11.0±0.25 12.0±0.58 RBC X 106/µL HGB (g/dL) 8.81±0.19 * 15.6±0.27 * 8.43±0.08 * 15.1±0.21 * 8.24±0.12 * 15.0±0.24 * 8.58±0.18 * 15.5±0.20 * 8.31±0.08 * 14.9±0.12 * 7.74±0.05 14.2±0.09 * 7.98±0.12 8.27±0.19 * 8.42±0.14 * 14.5±0.27 * 14.9±0.28 * 15.4±0.31 * 7.25±0.33 13.2±0.26 HCT (%) 56.3±1.25 * 54.0±0.54 * 54.4±1.17 * 54.6±0.83 * 52.6±0.46 * 50.8±1.25 * 50.0±1.25 * 51.2±1.33 * 54.2±1.35 * 43.9±2.15 MCV (fL) 63.9±0.75 64.1±0.55 66.0±0.87 * 63.7±0.70 63.3±0.71 65.6±1.64 * 62.7±0.41 61.9±0.52 64.3±0.64 60.8±1.27 27.8±0.21 * 28.0±0.37 * 27.6±0.37 * 28.4±0.50 28.3±0.16 28.1±0.79 28.9±0.69 29.2±0.31 28.4±0.21 30.0±0.43 PLT X 10 /µL 702±56.10 730±35.4 688±31.8 839±117 767±36.7 647±23.1 748±32.4 665±23.4 751±19.2 596±42.7 LYM (%) 78.4±4.74 73.8±1.85 69.3±1.52 72.6±2.60 69.5±1.96 72.8±7.69 72.8±11.1 77.4±1.78 74.6±1.46 78.9±1.12 NEUT (%) LYM # X 103/µL 21.6±4.74 8.54±0.48 26.2±1.85 8.26±0.59 27.4±2.60 * 10.2±1.16 30.5±1.96 * 9.20±0.94 27.2±7.69 8.40±1.82 27.3±11.1 * 7.73±1.06 22.6±1.78 6.32±0.87 25.4±1.46 * 8.18±0.35 21.1±1.12 9.47±0.90 NEUT#X 103/µL 2.42±0.58 3.97±0.85 3.98±0.17 2.80±0.40 4.70±3.20 1.84±0.27 2.78±0.14 2.53±0.19 2.90±0.11 M d te 3 30.7±1.52 * 4.98±0.53 * Ac ce p MCHC(g/dL) an EIML 100 2.20±0.26 PNML 300 PNML 1000 CONTROL ⃰ = p<0.05 (statistically significant); WBC – white blood cells; RBC – red blood cells; HGB – haemoglobin; HCT – haematocrit; MCV – mean corpuscular volume; MCHC - mean corpuscular haemoglobin concentration; PLT – platelets; LYM – lymphocytes; NEUT – neutrophils. 14 Page 14 of 23 us cr ip t Table 3: Effects of methanol extracts on general biochemical parameters of treated and untreated Wistar rats. Extracts and their concentration (mg/kg body weight) Parameters EIMB 100 89.5±3.31 PNML 100 85.7±5.20 PNML 300 83.0±2.50 PNML 1000 85.0±1.66 CONTROL 85.2±3.36 6.94±1.46 * 32.6±10.6 * 48.5±17.2 * 34.7±14.6 * 21.8±6.33 * 30.5±22.9 * 64.0±20.3 * 47.1±11.4 * 36.9±9.42 * 112±4.02 3.46±0.61 3.52±0.78 4.28±1.28 4.13±0.85 4.23±1.24 5.33±0.74 4.26±0.74 4.30±0.86 4.30±0.46 2.78±0.11 * 2.69±0.12 * 2.87±0.15 * 2.66±0.29 * 2.87±0.20 * 2.63±0.12 * 2.41±0.09 2.36±0.04 2.31±0.11 1.94±0.09 4.62±1.22 4.20±0.72 9.22±2.23 8.60±3.86 7.06±3.83 5.03±1.44 11.1±7.90 1.86±0.56 4.30±1.64 6.37±2.88 Creatinine (µmol/L) Cholestrol (mmol/L) HDL chol (mmol/L) 81.0±4.58 72.5±3.94 75.5±5.46 73.9±5.15 76.2±1.70 75.8±6.05 72.1±7.73 71.3±2.91 73.8±1.92 59.7±3.20 6.25±0.13 6.19±0.11 6.19±0.39 5.97±0.21 5.58±0.16 5.51±0.38 5.55±0.52 5.72±0.23 5.75±0.11 5.10±0.20 1.34±0.08 1.39±0.06 1.40±0.08 1.23±0.10 1.35±0.00 1.54±0.13 1.27±0.19 1.32±0.04 1.33±0.10 1.06±0.09 LDL chol (mmol/L) Glucose (mmol/L) Amylase (U/L) 4.60±0.09 4.50±0.09 4.48±0.26 4.52±0.11 4.03±0.13 3.70±0.28 4.03±0.31 4.15±0.17 4.30±0.07 3.58±0.16 0.48±0.11 * 0.33±0.04 * 0.40±0.07 * 0.40±0.09 * 0.39±0.05 * 0.25±0.02 * 0.43±0.11 * 0.35±0.04 * 0.33±0.01 * 1.17±0.28 803±55.3 769±34.80 837±35.50 807±11.6 848±35.2 809±67.3 785±12.0 751±19.80 828±7.77 810±71.50 d M EIML 1000 85.1±1.22 EIMB 300 82.5±1.50 an EIMB 1000 84.6±3.76 Tot protein (g/L) ALT/GPT(U/L) AST/GOT(U/L) Total bilirubin Gamma-GT(U/L) EIML 300 92.7±1.44 te EIML 100 92.8±2.11 5.52±0.53 Ac ce p ⃰ = p<0.05 (statistically significanct); ALT/GPT - Alanine aminotransferase; AST/GOT - Aspartate aminotransferase Tot protein – total protein; Gamma GT - Gamma glutamyl transferase; HDL chol - high density lipoprotein cholesterol; LDL chol - low density lipoprotein cholesterol 15 Page 15 of 23 4.0 Discussion Plants secondary metabolites are a wide range of molecules that have various pharmacological effects. These effects include therapeutic actions, defense mechanism for the plants and toxic effects on organs of animals and humans. The EIML, EIMB and PNML extracts revealed the presence of flavonoids, tannins, alkaloids and saponins (Table 1). With respect to biochemical analysis of the blood samples of the treated animals, there were us cr ip t significant increases (p<0.05) in the mean counts of red blood cells (RBC), haemoglobin (HGB) and hematocrit concentrations at all doses of administration of the extracts as compared to the untreated control group. Other studies have reported significant increases in these haematological parameters at 400, 800 and 1600 mg/kg dose levels of aqueous leaf extract of P. nigrescens (Agbor and Odetola, 2001) and at 50 and 100 mg/kg body weight of aqueous root extract of the same plant (Nsiah et al., 2006). This may support the folkloric use an of parts of this plant in Nigeria to treat anaemia. The EIMB extract showed increase in WBCs and lymphocytes (p>0.05) at dose of 100 M mg/kg. The reduction in the mean counts of WBCs (p>0.05) in rats administered with aqueous leaf extract of P. nigrescens has also been reported in previous reports (Agbor and Odetola, 2001; Nsiah et al., 2006). Owoyele et al. (2011) also reported decrease in mean d counts of WBCs in rats treated with lower doses of 50 and 100 mg/kg body weight of te aqueous root extracts of P. nigrescens. The active principles including phenols, cardiac effects. ep glycosides, terpenoids, saponins, alkaloids, tannins, and steroids may be responsible for these The various concentrations of the extracts increased the destruction of the erythrocytes or Ac c decreased its production or proliferation. Haemolytic activity provides the basic data and information on the interaction between compounds or extracts and biological agents at cellular level. Haemolytic activity of any compound or extract is an indicator of general cytotoxicity towards normal healthy cells (Da Silva et al., 2004). The presence of saponins in the extracts exhibit haemolytic activity in the cells by creating changes in the erythrocyte membrane (Kumar et al., 2011). Increase in these lipid profiles have been reported for P. nigrescens aqueous root extract at 100 and 150 mg/kg body weight by other studies. Increase in lipid profile such as HDL and LDL cholesterol in the Wistar rats may be useful indicators in investigating the influence of 17 Page 16 of 23 these extracts on metabolism of lipids and how animals and humans may be prone to coronary diseases from intake of preparations from these plants (Owoyele et al., 2011). The reduction in the blood glucose level (p<0.05) from all the doses of extracts administered may justify the folkloric use of P. nigrescens as an antidiabetic agent (Saba et al., 2010). E. ivorense should be investigated as a possible source of antidiabetic agent. us cr ip t Neutrophils are the predominant granulocytes that are seen in initial stages of acute inflammation (Alberts and Bruce, 2005). These neutrophils are packed with granules containing inflammatory factors like leukotrienes (Shen and Louie, 2005). These inflammatory factors could be involved in the evidence of inflammation shown in the micrograph of tissues (Fig. 4 and 5). These inflammations could cause a reduced flow of blood through kidneys (Rosner and Okusa, 2006). The result is a surge in blood creatinine an concentration (Table 3) and a decrease in the renal plasma clearance of creatinine (Guntupalli et al., 2006). Cheesbrough (1991) showed increase in serum creatinine levels of rats treated with aqueous leaf extract of Erythrophleum africanum and the associated reduction in renal M function. Since kidney and liver are organs of metabolism and excretion respectively of xenobiotic molecules such as saponins, alkaloids and tannins, the presence of these secondary d metabolites in E. ivorense may be responsible for the observed hepatorenal toxicities (Hassan te et al., 2007). The liver tissues of rats administered with EIML extract showed increased inflammatory ep infiltrates with few vacuolations and scarrings (cirrhosis), indicative of persistent damage while the EIMB extract treated rats showed liver tissue with profuse inflammatory cell Ac c infiltration and hepatocyte necrosis. The PNML extract at 1000 mg/kg body weight showed reduced inflammatory infiltrates with healthy hepatocytes and tissue proliferation, indicative of liver restitution (Fig. 5, G9). Alanine transaminase (ALT), aspartate transaminase (AST) and γ-glutamyl (GGT) are enzymes found in the cytoplasm of cells and they are involved in amino acid metabolism but only released into systemic circulation after cells have been damaged (Sallie et al., 1991; Burkill, 1995). Hassan et al. (2007) reported increases in serum levels of ALT and AST in treating rats with 2000 to 3000 mg/kg bwt of E. africanum extracts and suggested that the extracts must have affected the permeability of liver cell membranes and made them leaky, thus the leakage of ALT and AST to raise their serum levels. This was observed in the 18 Page 17 of 23 histopathology findings for the kidney and liver of animals treated with the various doses of the extracts with the exception of 100 mg/kg body weight of EIML (Fig. 4 and 5) which had no effect on both organs. An increase in the level of ALT and AST in blood serum of treated rats above normal ranges in the untreated rats may explain the liver damages by the methanol extracts (EIML, EIMB and PNML). t Spier et al. (1987) reported that hydrolysable tannins which are astringents bind to proteins in us cr ip plasma and body organs resulting in coagulation and necrosis. Blood from all the animals treated with the extracts (EIML, EIMB and PNML) showed increase in serum total protein concentration. Hassan et al. (2007) has also reported increases in the concentration of serum total protein in rats administered with extract of E. africanum. The increases in serum total protein concentration have been attributed to liver injury and hepatic toxicity (Gatsing et al., 2006; Emerson et al., 1993). The methanol extracts (EIML, EIMB and PNML) may therefore an be toxic to animals due to the measured increase in serum total protein concentration. The increasing doses of the extracts (EIML, EIMB and PNML) used in the study lead to increase M and pronounced toxicity in both livers and kidneys of all the treated animals. When cells are stressed, lysed or injured, they lose the integrity of their cytoplasmic d membrane. Lactate dehydrogenase (LDH) enzymes which are found in their cytosol can be te measured extracellularly as they leak through the disrupted cytoplasmic membrane. This study showed an increase in the amount of LDH released though not statistically significant ep (p>0.05) from the keratinocyte cells treated with concentrations of 1 to 10 µg/mL of the EIML and EIMB extracts and 1 to 100 µg/mL of the PNML extracts as compared with the untreated cells (negative control). Triton X-100 is a cell lysing agent and thus HaCaT Ac c keratinocytes treated with it showed marked release of LDH enzymes, evident of pronounced cell damage but not as compared to the extract treated cells. The synergistic effects of plant metabolites may sometimes be toxic and cause damage to cells treated with them. The methanol extracts of these plant materials may thus possess compounds that may be toxic to morphology and function of cells of humans and animals. This cytotoxic property may support the overall in vivo toxicity observed in the kidney and liver sections of the Wistar rats. There was reduction in the release of LDH as the concentration of the extracts increases and this may be due the presence of bioactive agents in the extracts which in higher concentrations may serve as protective mechanism for the cells or delay or reduce apoptosis. 19 Page 18 of 23 There is a need to identify and characterize the individual compounds or agents and the subsequent protective mechanism to cell damage by higher concentrations of the extracts. Conclusion Methanol leaf and bark extracts of E. ivorense and leaf and other aerial part extract of P. nigrescens may exhibit dose and time dependent toxicity to animals and humans. The kidney t and liver tissues from rats administered with the extracts showed diseased conditions us cr ip including inflammation of cells, necrotic tissues and infiltration cells. The extracts exhibited cytotoxicity on HaCaT keratinocytes at low concentrations but not statistically significant. Acknowledgements We are grateful to Dr. Alex Asase and Mr. John Yaw Amponsah of the Department of Botany, University of Ghana, Accra and Mr. Eric Gyebi, for the identification and collection an of the plant materials. We thank Mr. Thomas Ansah, Department of Pharmacology for the technical assistance in the animal studies. We thank Prof. Dr. Andreas Hensel, Institute for Pharmaceutical Biology and Phytochemistry, University of Muenster, Germany for the M cytotoxicity studies. 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