Globimetula braunii, a major medicinal plant used in traditional medicine in Nigeria, was
evaluated for possible effects on hepatic and hematologic indices in Wister albino rats. The crude methanol extract of the plant was successively fractionated with hexane, chloroform, ethyl acetate, butanol and water. The MeOH extract of G. braunii and its fractions were administered to albino rats at a dose of 200 mg kg-1 body weight for 14 days. Assessments of liver function enzymes aspartate amino transferase (AST), alanine amino transaminase (ALT), as well as total and direct bilirubin levels were carried out. We observed significant increases (p≤0.05) in packed cell volume (PCV) and hemoglobin (Hb) in the rats treated with chloroform extract (CHCl3), ethyl acetate (EtOAc) and water (H2O) fractions. The red blood cell (RBC) count increased (p<0.05) after administration of
CHCl3 and EtOAc fractions while white blood cell (WBC) count increased (p<0.05) in the
crude and all its fractions except butanol. Significant decreases (p0.05) in enzyme activities in the MeOH fraction. However, the administration of other
fractions to the rats, led to increases (p<0.05) in the activities of both enzymes. Total and direct bilirubin showed increases (p<0.05) in EtOAc and hexane fractions while only direct bilirubin increased (p<0.05) in the crude fraction. The present study demonstrates that G. braunii chloroform fraction has an influence on hematologic
functions and liver enzyme levels in rats

Kareem, G.K.

Ogbunugafor, H.A.

Okpuzor, J.

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EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  182 Original article:  HEPATIC AND HEMATOLOGIC EFFECTS OF FRACTIONS OF  GLOBIMETULA BRAUNII IN NORMAL ALBINO RATS   J. Okpuzor1.*, H. A. Ogbunugafor2, G. K. Kareem1   1 Department of Cell Biology and Genetics, University of Lagos, Akoka – Lagos, Nigeria 2 Department of Applied Biochemistry, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria * Corresponding author: Email: joyokpuzor@yahoo.com; Telephones: +234 802 306 5999, +234 803 361 1553  ABSTRACT  Globimetula braunii, a major medicinal plant used in traditional medicine in Nigeria, was evaluated for possible effects on hepatic and hematologic indices in Wister albino rats. The crude methanol extract of the plant was successively fractionated with hexane, chloroform, ethyl acetate, butanol and water. The MeOH extract of G. braunii and its fractions were ad-ministered to albino rats at a dose of 200 mg kg-1 body weight for 14 days. Assessments of liver function enzymes aspartate amino transferase (AST), alanine amino transaminase (ALT), as well as total and direct bilirubin levels were carried out.  We observed significant increases (p≤0.05) in packed cell volume (PCV) and hemoglobin (Hb) in the rats treated with chloroform extract (CHCl3), ethyl acetate (EtOAc) and water (H2O) fractions. The red blood cell (RBC) count increased (p<0.05) after administration of CHCl3 and EtOAc fractions while white blood cell (WBC) count increased (p<0.05) in the crude and all its fractions except butanol. Significant decreases (p<0.05) in the activities of AST and ALT enzymes were observed in rats treated with chloroform fraction, while there was elevation in the activity of ALT in the butanol fraction group. There was no difference (p>0.05) in enzyme activities in the MeOH fraction. However, the administration of other fractions to the rats, led to increases (p<0.05) in the activities of both enzymes. Total and di-rect bilirubin showed increases (p<0.05) in EtOAc and hexane fractions while only direct bilirubin increased (p<0.05) in the crude fraction. The present study demonstrates that G. braunii chloroform fraction has an influence on hema-tologic functions and liver enzyme levels in rats.   Keywords: Globimetula braunii, medicinal plant, fractions, hepatic, hematologic, toxic   INTRODUCTION  The use of medicinal plants for treat-ment and management of diseases has been gaining prominence world wide especially in the developing countries where 80 % of the population still depends on traditional healing methods (Iwu, 1993; De Silva, 1997). These plants are used primarily for the treatment of numerous diseases like hy-pertension (Adjanohoun et al., 1985), car-diovascular diseases (Ouedraogo et al., 2004), hepatic illness (Phillipson & Wright, 1991), arthritis (Dongmo et al., 2003) dia-betes (Al-Ghaithi et al., 2004), malaria (Traore et al., 2000). This surge in the use of herbal medicines is probably due to the perceived failure of synthetic drugs in the treatment of some chronic diseases like hy-pertension, diabetes, arteriosclerosis etc; the side effects associated with most drugs and the incidence of drug resistance especially EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  183 among the antibiotics family.  Globimetula braunii Engl van Tiegh (Loranthaceae) is a hemi-parasitic shrub that grows on dicotyledonous trees and at-taches itself to the host by modified roots (Burkhill, 1985). The plant is widely dis-tributed in tropical African countries such as Ghana, Cameroun and Nigeria and is a major ingredient used in herbal medicines. G. braunii commonly called “Afomo onis-hano” by the Yoruba tribe of south west Nigeria has gained importance as being use-ful in the treatment of headache, rheumatic pains and pulmonary troubles (Burkhill, 1985). Leaves, fruits and flowers of the plant are used to treat hypertension, while the roots are employed for other therapeutic uses such as ulcer and cancer treatment (Burkhill, 1985). The liver plays a central role in metabo-lism of drug and xenobiotics, protein syn-thesis and in maintaining biologic equilib-rium of organisms. Due to these important roles, liver enzymes are used as markers in assessment of drug or plant extract safety or toxicity (Tietz, 2000; Satyapal et al., 2008). The transaminases are involved in interme-diary metabolism and are thus present in high concentration in the liver (Tietz, 2000). They are rapidly released into the serum in cases of acute destruction of tis-sues as in myocardiac infarction or hepato-cellular necrosis (Tietz, 2000). Therefore, the extensive use of this plant underscores the need to evaluate its toxic and hematologic effects. Our search on the previous use of the plant did give an insight on previous toxic and hematologic properties of the leaf extract and fractions of G. braunii. Thus, the aim of this study is to evaluate the effect of the methanol ex-tract of the leaf and fractions of G. braunii on liver and blood parameters in rats.  MATERIALS AND METHODS  Plant material The leaves of Globimetula braunii were collected in March 2008 from Olokemeji forest reserve in Ogun State, Nigeria. They were authenticated at Forestry Research Institute (FRIN) Ibadan, Nigeria and a specimen was prepared and deposited at the Institute’s herbarium (FHI107741).   Animal’s source and sorting Thirty five adult Wistar albino rats were purchased from the animal colony of the Department of Biochemistry, University of Ibadan, Ibadan, Nigeria. The rats were al-lowed to acclimatize for two weeks in the animal house of University of Lagos. Ap-proval for the use of the animals was ob-tained from the Ethical Committee of Uni-versity of Lagos for experimental purposes only. The rats weighing between 180-200 g were sorted into 7 groups of 5 rats each. They were housed in standard rat cages un-der standard laboratory conditions and al-lowed feed and water ad libitum.   Chemicals Assay kits for Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) were from Randox Laboratories Ltd, Ard-more, UK and the kit for bilirubin determi-nation was from Human Gesellschaft für Biochemica und Diagnostica, Wiesbaden, Germany. They were purchased from Nzemat Pharmaceuticals Company, Fola-Agoro, Lagos, Nigeria. All other reagents and solvents used were of analytical grade.   Extraction of plant The dried leaves of Globimetula braunii were pulverized into a fluffy mass. 303 g of the powdered leaves was extracted with 250 ml of 80 % MeOH using Soxhlet ex-tractor for 8 hours. The extract solution was evaporated to dryness under reduced pres-sure (below 40 °C) to yield 35.88 g crude extract.    Fractionation of the extract The method described by Yesilada and Kupeli (2002) was employed. The metha-nolic extract of G. braunii was reconstituted with 200ml of a mixture of MeOH:H20 (9:1) and shaken with hexane (3 x 100 ml). Combined hexane fraction was evaporated under reduced pressure to yield “hexane EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  184 fraction” (4.95 g). MeOH was evaporated from the remaining extract and diluted with distilled water to 200 ml and further frac-tionated by successive solvent extraction with chloroform (CHCl3) (4 x100 ml), ethyl acetate (EtOAc) (2 x 100 ml) and n-butanol saturated with H20 (3 x 100 ml). Each ex-tract was evaporated to dryness under re-duced pressure to yield CHCl3 fraction (4.29 g), EtOAc fraction (2.87 g), butanol fraction (1.59 g) and remaining H20 fraction (3.86 g).   Preparation of extract and fractions solu-tions The extract and fraction solutions were prepared by dissolving 0.4 g of the extract and fractions in 10 ml of 2 % Tween 80, to give an effective concentration of 40 mg mL-1. The formula: Dosage mg kg-1 /1000 x Wt of animal (g)/concentration (mg mL-1) was used to calculate the volume of the ex-tract/fractions solution to be administered to each animal. The solutions were prepared fresh daily before administration.   Animal treatment The animals were distributed into seven groups consisting of five rats per group. One milliliter of the crude and different fractions were administered orally at a dose of 200 mg kg-1 body weight to the test groups for 14 days. The control group re-ceived 1 ml of 2 % Tween 80 only. The animals were fasted overnight, sacrificed and blood was collected. The blood for he-matologic studies was stored in EDTA bot-tles. The blood for biochemical studies was left to coagulate and centrifuged at 3000 rpm for 10 min; and serum was sepa-rated.   Determination of hematologic parameters Hematologic parameters were deter-mined by standard methods. Packed cell volume (PCV) was determined by micro-hematocrit method as described by Dacie and Lewis (1991, 1995) using microhema-tocrit centrifuge (Uniscope Sm 112, Spring-field Medicals, U.K.). Hemoglobin concen-tration was determined by the Cyanome-themoglobin method (Jain, 1986). The red cells (RBC) and white blood cells (WBC) were estimated using improved Neubauer counting chamber (Dacie & Lewis, 1991, 1995).   Determination of biochemical parameters Assay of Alanine amino transaminase (ALT) and Aspartate amino transaminase (AST) was carried out by the procedure outlined in the diagnostic kit as described by Reitman and Frankel (1957). Total and direct bilirubin was determined by proce-dure outlined in the diagnostic kit as de-scribed by Jendrassik and Grof (1938).  RESULTS  Fractions of Globumetula braunii were administered to experimental rats to evalu-ate their toxic and hematologic effects. Re-sults revealed significant increases (p<0.05) in PCV (%) values and RBC levels in CHCl3, EtOAc and H2O fractions (Figures 1 & 2) while there were no significant changes in the levels of these two parame-ters in methanol, hexane and butanol frac-tions. The Hb concentration in chloroform, ethyl acetate and water fractions (Table 1) showed significant increase p≤0.05 but no significant increases were evident in metha-nol, hexane and butanol fractions compared to control.  Table 1: Effect of fractions of G. braunii on Hb concentration (mg/dL) in rats  Groups Hemoglobin concentration (mg/dL) Control 8.50 ± 0.11 MeOH 10.18 ± 1.01 Hexane 11.01 ± 1.04 CHCl3 12.72 ± 0.70* ETOAc 10.59 ± 0.42* Butanol 9.11 ± 0.39 H2O 10.53 ± 0.63* *significant at p≤0.05  Furthermore, there were significant in-creases (p<0.05) in WBC (cells/mm3) in all the fractions except in butanol fraction (Figure 3).EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  185 051015202530354045Control MeOH extract Hexane CHCl3 EtOAc Butanol H2OPCV (%) Figure 1: The effect of fractions of G. braunii on PCV level in albino rats  00.511.522.533.544.55Control MeOH extract Hexane CHCl3 EtOAc Butanol H2ORBC (cell/mm^3)x10^6 Figure 2: The effect of fractions of G. braunii on RBC in albino rats  EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  186 020004000600080001000012000140001600018000Control MeOH extract Hexane CHCl3 EtOAc Butanol H2OWBC (cells/mm^3)x10^6 Figure 3: The effect of fractions of G. braunii on WBC in albino rats  The direct bilirubin levels significantly elevated (p<0.05) in the methanol, hexane and ethyl acetate fractions while total bilirubin significantly increased in the hex-ane, ethyl acetate and water fractions only (Table 2). However, both hexane and ethyl acetate fractions showed significant in-creases in both total and direct bilirubin levels.   Table 2: Effect of fractions of G. Braunii on se-rum bilirubin concentration (µmol/L) in rats  Groups Direct bilirubin (µmol/L) Total bilirubin (µmol/L) Control 0.58 ± 0.03 1.32 ± 0.004 Crude 0.85 ± 0.06* 1.70 ± 0.14 Hexane 0.67 ± 0.02* 1.49 ± 0.06* CHCl3 0.63 ± 0.03 1.36 ± 0.07 ETOAc 1.16 ± 0.04* 2.37 ± 0.13* Butanol 0.60 ± 0.04 1.39 ± 0.04 H2O 0.59 ± 0.02 1.46 ± 0.034** significant at p<0.05  The assay of the hepatic enzymes AST and ALT in Figure 4, revealed that serum AST and ALT decreased (p<0.05) in chlo-roform fraction while ALT activity in-creased (p<0.05) in hexane, EtOAc, butanol and H2O fractions. AST activity also in-creased (p<0.05) in all the fractions except butanol. There was no significant change (p>0.05) in the activities of these enzymes in the methanol fraction.  DISCUSSION  Different solvent systems are routinely used to identify different bioactive compo-nents from plant extracts. The results of this investigation demonstrate that hydrophobic and hydrophilic solvents isolated different active principles which exhibited different hematologic and biochemical effects in rats.  The ability of some fractions (CHCl3, EtOAc and H2O) to increase the PCV level in albino rats is an indication of hemocon-centration and may be due to increased RBC mass. Increased RBC is suggestive of polycythemia and a positive erythropoetic effect. Thus, it is likely that these fractions enhanced the oxygen carrying capacity of the animals. This is supported by other workers that reported similar result on ethanol stem-bark extract study of Mangif-era indica L. and Garcinia cambogia (Oluyemi et al., 2007; Nwinuka et al., 2008). Furthermore, the increased WBC count indicates that these fractions to an extent, affected the defense mechanism of treated rats (Oluyemi et al., 2007). EXCLI Journal 2009;8:182-189  ISSN 1611-2156 Received: July 11, 2009, accepted: August 01, 2009, published: August 04, 2009  187 05101520253035Control MeOH extract Hexane CHCl3 EtOAc Butanol H2OEnzyme Activity (IU/L)ALTAST Figure 4: The effect of fractions of G. braunii on ALT and AST activity in albino rats  The elevated bilirubin levels may sug-gest lyses of red cells in EtOAc fraction-treated rats, and agrees with our earlier studies of this fraction of G. braunii where increased MDA levels were recorded as a result of elevated lipid peroxidation (Okpu-zor et al., 2009). This is likely due to loss of functional integrity of the cellular mem-brane of the red cells in the EtOAC frac-tion-treated rats (Iweala & Okeke, 2005). The marked difference in the effect of the G. braunii fractions on liver enzymes ALT and AST, support the view of additive and supra-additive action of a plant’s multiple constituents as stated by Spinella (2002). This synergy is demonstrated in the crude fraction of G. braunii which exhibited no significant change in ALT and AST levels while the reduction in the levels of these enzymes in the chloroform fraction may indicate the healing potential and non toxic-ity (Scott Luper, 1998; Smith et al., 2002). However, the increased ALT and AST ac-tivities in the other fractions may suggest that this dose is toxic and damaging to the liver. This is supported by other workers who have reported that some plant extracts are hepatotoxic (Gathumbi et al., 2000; Shahraki et al., 2007).  In conclusion, this study demonstrates that the chloroform fraction contains bioac-tive agents that influence hematologic pa-rameters and levels of liver enzymes in rats.   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Hepatic and hematologic effects of fractions of Globimetula Braunii in normal albino rats

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Hepatic and hematologic effects of fractions of Globimetula Braunii in normal albino rats

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