THE EFFECT OF VERNONIA CALVOANA EXTRACT ON SOME HAEMATOLOGICAL INDICES IN ACETAMINOPHEN TREATED MALE WISTAR RATS

The effect of Vernonia calvoana extract on some haematological indices in acetaminophen (paracetamol) treated albino Wistar rats was investigated in this study. Thirty-five (35) albino Wistar rats weighing 100-150g were randomly assigned into five (5) groups of seven rats each. Group 1 was the normal control ( Normal saline), group 2 received normal saline after treating with 2g/kg Acetaminophen (Paracetamol) via oral gavage, group 3 was treated with 200mg/kg body weight of V.C, group 4 received 400mg/kg body weight of V.C and group 5 was administered 100mg/kg body weight of Vitamin E. All treatments were done orally gavaged and lasted for twenty-one days. At the end of the treatment period, animals were euthanized using chloroform vapours, and blood was collected via cardiac puncture. The blood was collected and used for evaluation of some haematological indices. Results indicated significant (p<0.001) increase in red blood cell count, haemoglobin and packed cell volume in the 400mg/kg body weight Vernonia calvoana extract treated group compared with acetaminophen treated group. Similarly, platelet count, Lymphocytes and white blood cell count were markedly increased in the 200mg/kg VC, 400mg/kg VC and vit Etreated groups (p<0.05). We, therefore, conclude that ethanolic leaf extract of Vernonia calvoana administered at 400mg/kg body weight possess anti-anaemic properties.


INTRODUCTION
Acetaminophen (Paracetamol -PCM) is one of the most commonly found drugs in a lot of homes. It is the most widely used and available analgesic in Nigeria. As a result, there is very high potential for abuse and an eventual overdose of the drug (Sheen et al., 2002). Paracetamol toxicity due to overdose is one of the most common causes of poisoning worldwide (Gunnell, et al., 2000). Acute paracetamol overdose can result in potentially dangerous liver and kidney failure in both human and experimental animals and may result in death in extreme cases (Lorz et al., 2004). The kidney happens to be the second target organ of paracetamol toxicity, though nephrotoxicity may exist independent of hepatoxicity following overdose (Gulnaz, et al., 2010).
The role of kidney in erythropoiesis cannot be over emphasised. Most ethnobotanicals has been reported to boost haemoglobin levels in animals It, therefore, becomes imperative to investigate the possible haematopoietic activity of Vernonia calvoana leaf extract in acetaminophen-induced Wistar rats.
Renal impairment is directly associated with quite a number of haematopoietic changes/distortions. There is a strong correlation between anaemia and the degree of renal dysfunction, with the major cause being the failure of kidneys to produce erythropoietin (Alpers, 2004). Anaemia is referred to as an ordinary complication of chronic kidney disease or chronic renal failure (progressive decreased loss of renal function) because, due to damage or loss of function of the kidney, the epithelial cells cannot efficiently produce the erythropoietin required for erythropoiesis to take place. This will result in the reduction of red blood cell (RBC) count (Hodges et al., 2007). Erythropoietin is a hormone produced by epithelial renal cells. It regulates red blood cell production and assists in the maintenance of the viability and integrity of the cells (Suresh et al., 2012) Medicinal plants have played an important role in the abatement of toxic substances in the human body. They also function as vital hypolipidemic agents (Luo et al., 2004). There are so many plants with potent medicinal properties in the tropics, with Vernonia being a common genus of such plants. Tende et al. (2013) reported the non erythropoietic effects of Vernonia amygdalina extracts in Wistar rats. The most commonly eaten ones are Vernonia calvoana, Vernonia amygdalina, with the former being the plant of interest. Vernonia calvoana belongs to the Asteraceae family. It is commonly called "Ekeke leaf" and "Uchu nyin" respectively by the indigenes of the central and northern senatorial districts of Cross-River State of Nigeria (Igile et al., 2013, Egbung et al., 2016. The emphasis on Vernonia amygdalina has resulted in the neglect of other less utilised species that may have some ethnomedicinal properties. Vernonia calvoana has been sparsely investigated compared to other Vernonia species. The anti-oxidant properties of the leaves as well as the inflorescents of Vernonia calvoana has been established (Igile et al., 2013, Egbung et al., 2016. However, there is little or no information on the erythropoietic potentials of the extract of Vernonia calvoana in acetaminophen treated Wistar rats. The present study was designed to fill this information gap.

Preparation of ethanolic extract
The leaves were washed thoroughly to remove dust and other forms of dirt and afterwards air dried at room temperature (27 0 C ± 2 0 C) for seven days to remove moisture until completely dry. The dried leaves were blended to a fine powder using a dry Moulinex super blender and stored in air-tight containers. 1.5kg of the powder was weighed using an electronic scale and afterwards soaked in 2000ml of methanol and dichloromethane (1:1, v/v) in a ratio of 3:4, i.e. (powder/solvent). To allow for proper mixing of the powder and the solvent, the mixture was put in the blender, agitated and then put in air-tight containers. The containers holding the mixtures were kept in the refrigerator at a temperature of 4 0 C for 48 hours. Filtration of the mixture was accomplished first by using a cheese cloth, followed by the Whatman no.1 filter paper (24cm). The filtrate was concentrated using a rotary evaporator (model RE52A, China) to 10% of its original volume at a temperature of 37 0 C -40 0 C. It was then concentrated to complete dryness in a water bath. The extract was afterwards refrigerated at 2-8 0 C until when required for administration.

Experimental animals and design
Thirty-five albino Wistar rats comprising of both sexes weighing between 100 to 150g, were obtained from a disease-free stock of the animal house, Department of Zoology, University of Calabar, Calabar. The animals were acclimatized for two weeks on pelletized rat chow and water provided ad libitum and were maintained under standard housing condition of adequate ventilation and room temperature of 28 0 C ± 2 0 C and relative humidity of 46% ± 5% and a natural 12 hour light -dark regimen in the Biochemistry Department's animal house, where they were kept for the period of the experiment. Permission and approval for the use of the animals to carry out the study according to approved protocol was obtained from the Faculty of Basic Medical Sciences Ethical Committee, University of Calabar, Calabar. The animals were weighed and assigned randomly to five groups of seven rats each based on weight. All the groups were allowed free access to pelletized rat chow and clean water. See experimental design below:

Extract and drug administration
The doses (200 mg/kg and 400 mg/kg) used were based on the predetermined LD 50 values obtained using the method of Lorke (1983).The dose of 2g/kg bwt of paracetamol was based on the report of Suchismita et al. (2015). The extract was diluted in normal saline, which acted as the vehicle and administered orally through gastric intubation accordingly. The control animals received 0.2ml of normal saline. Treatment period with the Vernonia calvoana leaf extract lasted for a period of 21 days. The animals were fasted 12 hours overnight prior to the time of sacrifice. The animals were euthanised with chloroform and blood samples collected via cardiac puncture.

Haematological indices determination
Whole blood was collected from each experimental animal through cardiac puncture and put into to sterile Ethylenediamine tetra acetic acid, sample tubes and used for determination of haematological indices (Haemoglobin, red blood cell, white blood cell, platelet and packed cell volume auto analyzer was used for the estimations.

Statistical Analysis
Data obtained were analysed using o analysis of variance and differences between pairs of groups were determined using posthoc comparisons. Results were expressed as mean ± standard error of mean (SEM). Data were considered significant at p<0.05, p<0.01 and p<0.001. Computer software SPSS version 17.0 and Microsoft excel (2007 version) were used for analysis. Group 2 shows a significant decrease at p level compared to group 1. Also, the Hb. level group treated with 400mg/kg bwt of the extract (group 4) shows a significant increase at p < 0.05 compared to group 2 (nephrotoxic untreated group). Vitam group (group 5) revealed a significantly lower Hb. concentration compared to the normal control 0.05 and a lower Hb. level compared to group 4 at 0.05. The packed cell volume (PCV) result ( showed a significant (p < 0.001) decrease in compared to group 1. Similarly, group 3 indicated lower PCV value, significant at p < 0.01 compared normal control as well. There was a significant

Results
Results of the haematological effects of Vernonia calvoana extracts in Acetaminophen treated Wistar rats are presented in figures 1 untreated nephrotoxic group indicated lower mean RBC count at p < 0.001, compared to the normal control group. The group treated with 200 bwt of the extract had a slightly lower RBC count, significant at p < 0.01 compared to the normal control. The group treated with 400 mg/kg of the extract showed a significantly higher RBC count compared to the untreated nephrotoxic group at p comparison with the group treated 200 400 mg/kg bwt treated group showed a significant increase in RBC count at p < 0.01, indicating a dependent increase. The vitamin E treated group showed a significantly lower RBC count compared to the normal control and the group treated 400 < 0.01 respectively.
extract on Red blood cell count in paracetamol treated rats. Values are mean ±SEM, n = 5.**p<0.01,***p<0.001 vs control, a = p<0.001 vs N.untreated, e = p<0.01 vs N + LD V.C, y = p<0.01 vs N + HD V.C shows a comparison of haemoglobin across the experimental groups. < 0.05 in Hb o group 1. Also, the Hb. level of the of the extract (group 4) 0.05 compared to p 2 (nephrotoxic untreated group). Vitamin E treated a significantly lower Hb. ared to the normal control at p < pared to group 4 at p < result (Figure 3) 0.001) decrease in group 2 roup 1. Similarly, group 3 indicated a 0.01 compared to the There was a significant increase in PCV at p<0.001 in group 4 when compared to group 2. A dose-dependent increase in PCV was observed group 4 showed a significant increase in PCV at p 0.001 compared to group 3. PCV of group 5 wa significantly lower at p < 0.001 compared to the normal control and group 4 respectively. Treatment with 400mg/kg body weight V.C indicated a significantly higher value of lymphocytes at p < 0.001 relative to group 2 (Figure 4 lower lymphocyte count significant at p to the normal control and a significantly higher value at p < 0.01 as well when compared to paracetamol group (group 2). Group 5 shows a significantly lower value at p < 0.001 relative to the control as well as group 2 and 4 at p < 0.01

EXTRACT ON SOME HAEMATOLOGICAL INDICES
Results of the haematological effects of extracts in Acetaminophen (PCM)rats are presented in figures 1 -6. The untreated nephrotoxic group indicated a significantly 0.001, compared to the normal control group. The group treated with 200 mg/kg of the extract had a slightly lower RBC count, 0.01 compared to the normal control. mg/kg of the extract bwt ntly higher RBC count compared to the untreated nephrotoxic group at p < 0.001. In comparison with the group treated 200 mg/kg bwt, the treated group showed a significant 0.01, indicating a doseincrease. The vitamin E treated group showed a significantly lower RBC count compared to the normal control and the group treated 400 mg/kg bwt at p 0.01 respectively.
ed rats. Values are mean ±SEM, n = 5.**p<0.01,***p<0.001 vs control, a = p<0.001 vs N.untreated, e = p<0.01 vs N + LD V.C, y = p<0.01 vs N + HD V.C when compared to group increase in PCV was observed a significant increase in PCV at p < red to group 3. PCV of group 5 was 0.001 compared to the normal Treatment with 400mg/kg body weight V.C indicated a significantly higher value of lymphocytes at p Figure 4). Group 3 had a lower lymphocyte count significant at p < 0.01 compared ontrol and a significantly higher value at p 0.01 as well when compared to paracetamol -induced Group 5 shows a significantly lower 0.001 relative to the control as well as group 2 and 4 at p < 0.01 The platelet counts (figure 5) showed significant (p < 0.001) decrease in the paracetamol induced compared to the normal control. A significant (p < 0.001) increase in platelet count was observed in group 3 relative to group 2. The 400 mg/kg V.C treated group indicated a significantly increased platelet count at ) showed a in the paracetamol-. A significant (p ncrease in platelet count was observed in The 400 mg/kg V.C treated a significantly increased platelet count at (p < 0.001) compared to the normal contr respectively. The same pattern was observed when compared to group 3. Group 5 showed higher platelet count at p < 0.05 groups 1 and 2 respectively extract on platelet count in paracetamol treated rats. Values are mean ±SEM, n = 5. *p<0.05,***p<0.001 vs control, a = p<0.001 vs N. untreated, d = p<0.001 vs N + LD V.C, x = p<0.001 vs N + HD V.C EXTRACT ON SOME HAEMATOLOGICAL INDICES extract on lymphocyte count in paracetamol treated rats. Values are mean ±SEM, n = 5.
untreated, e = p<0.01 vs N + LD V.C, x = p<0.01 vs N + 0.001) compared to the normal control and group 2 respectively. The same pattern was observed when compared to group 3. Group 5 showed a significantly 0.05 when compared to s 1 and 2 respectively.
in paracetamol treated rats. Values are mean ±SEM, n = 5. *p<0.05,***p<0.001 vs control, a = p<0.001 vs N. untreated, d = p<0.001 vs N + LD V.C, x = p<0.001 vs N + HD V.C The white blood cell count ( Figure 6 untreated nephrotoxic group (group 2) had a significantly lower WBC count at p < 0.001 compared with th normal control. The WBC count of the group treated with 400 mg/kg bwt of the extract increased significantly at p < 0.05 compared to the normal control and group 2

DISCUSSION
Acetaminophen (paracetamol) has been reported to exert toxicity in Wistar rats at 2000mg/kg bwt or 2g/kg bwt (Suchismita et al., 2015).The untreated nephrotoxic group (group 2) that received 2g/kg showed a decrease in red blood cell count, Hb concentration, PCV, and platelet count compared to the normal control and the Vernonia calvoana treated groups. A strong correlation has been established between anaemia and the degree of renal dysfunction, with the major cause being the failure of the kidneys to produce erythropoietin (Alpers, 2004). Renal impairment has been linked directly to a number of haematological distortions. Therefore, the reduced red blood cell count could be as a result of a compromised erythropoietin production as a result of damage acetaminophen overdose. Erythropoietin implicated in the humoral regulation of the platelet mass (Suresh et al., 2012). However, the group treated with 400 mg/kg bwt of the extract showed an improvement in erythrocyte count, Hb concentration, PCV and platelet count. This indicates a positive haematopoiet that is contrary to the findings of Tende et al investigated a sister specie, Vernonia amygdalina Wistar rats. The haematopoietic activity of calvoana extracts which is comparable to Vitamin E treated group may be attributed to the proximate/chemical composition of the plant the flavonoids. Vitamin E is a synthetic antioxidant drug 260 GODWIN ENEJI EGBUNG, ITEM JUSTIN ATANGWHO, OCHUOLE DIANA ODEY AND SUNDAY AGBA BISONG hite blood cell count (Figure 6) in the a significantly compared with the . The WBC count of the group treated with increased significantly at p d to the normal control and group 2 respectively. Similarly, the group trea (group 5) had a higher WBC count significant at p relative to group 1. WBC count of group 5 also increased significantly at p < 0.001 compared to group 2 and 3 extract on the white blood cell count in paracetamol treated rats. Values are mean ±SEM, n = 5. *p<0.05,**p<0.01 vs control, a = p<0.001 vs N. untreated, e = p<0.001, d = p<0.001 vs N + LD V.C

Acetaminophen
(paracetamol) has been rats at 2000mg/kg bwt The untreated 2g/kg of PCM showed a decrease in red blood cell count, Hb and platelet count compared to the calvoana extract treated groups. A strong correlation has been established between anaemia and the degree of renal dysfunction, with the major cause being the failure of the 2004). Renal en linked directly to a number of haematological distortions. Therefore, the reduced red compromised as a result of damage due to Erythropoietin is also of the platelet mass However, the group treated with of the extract showed an improvement in erythrocyte count, Hb concentration, PCV and platelet count. This indicates a positive haematopoietic effect et al. (2013) that Vernonia amygdalina in . The haematopoietic activity of Vernonia which is comparable to Vitamin E attributed to the composition of the plant especially Vitamin E is a synthetic antioxidant drug that prevents oxidative stress induced damage in tissues of living organisms.
Igile et al., appreciable amounts of protein, vitamins, folic acid an minerals. Earlier reports also indicated diets have the ability to increase PCV and Hb concentration in human and animal studies ( et al., 1991, Alada et al., 2004 The lymphocyte and white blood cell count of the nephrotoxic untreated group decreased significantly. This agrees with a human study report of (Arun 2012), where patients who suffered from a progressive deterioration of their renal function experienced decline in their immune status, following a decrease in white blood cell count. The precise mechanism of action behind this reduction has not been fully understood. The groups treated with the extract showed a dependent increase in lymphocyte and The leucocyte count increase could be attributed to the presence of zinc in the plant in appreciable amounts, which is capable of stimulating the formation of white blood cells according to (Claude and Paule increase in lymphocyte count could also imply possible immune boosting potentials since lymphocytes are known to have the ability to fight diseases.

CONCLUSION
From the results obtained in this study, it is safe to further confirm that the ethanol ITEM JUSTIN ATANGWHO, OCHUOLE DIANA ODEY AND SUNDAY AGBA BISONG Similarly, the group treated with vitamin E a higher WBC count significant at p < 0.01 to group 1. WBC count of group 5 also 0.001 compared to groups respectively. treated rats. Values are mean ±SEM, n = 5. *p<0.05,**p<0.01 vs control, a = p<0.001 vs N. untreated, e = p<0.001, d = p<0.001 vs N + LD V.C that prevents oxidative stress induced damage in tissues et al., (2013) reported nts of protein, vitamins, folic acid and minerals. Earlier reports also indicated that protein-rich diets have the ability to increase PCV and Hb concentration in human and animal studies (Bolarinwa Alada, 2000). lymphocyte and white blood cell count of the nephrotoxic untreated group decreased significantly. This agrees with a human study report of (Arun et al., 2012), where patients who suffered from a progressive deterioration of their renal function experienced a ollowing a decrease in count. The precise mechanism of action behind this reduction has not been fully understood. The groups treated with the extract showed a doseincrease in lymphocyte and leukocyte count. The leucocyte count increase could be attributed to the presence of zinc in the plant in appreciable amounts, which is capable of stimulating the formation of white Claude and Paule, 1979). The te count could also imply possible since lymphocytes are known to have the ability to fight diseases.
From the results obtained in this study, it is safe ethanol leaf extract of