Evaluation of haematological and plasma biochemical effects of aqueous extracts of Parkia biglobosa seeds in rats

The effects of sub-acute exposure to aqueous extract of Parkia biglobosa seeds on haematology and plasma biochemical parameters were studied. Twenty eight male Wistar albino rats were orally ingested with 0, 200, 400 and 800 mgkg -1 dose of the extract for 21 days and were sacrificed. Haematological parameters were assessed: red blood cell counts, haematocrit and haemoglobin concentration significantly (p<0.05) increased at the 800 mgkg -1 dose. We also observed significant increases in white blood cells, lymphocytes and neutrophils at this dose. Evaluation of effect of seed extract on plasma biochemical values revealed significant increase in total protein, albumin and aspartate amino transferase. However, the extract caused significant decrease (p < 0.05) in the levels of blood urea nitrogen and creatinine. Changes observed in plasma levels of globulin, alanine amino-transferase (ALT) and gamma-glutamyl transferase (GGT) were not significantly different from the control at all treatment doses.


INTRODUCTION
The African locust bean tree, Parkia biglobosa, is a perennial tree legume, belonging to the sub-family mimosoidae and family leguminosae.The plant has been used as a source of food, medicinal agents, timber and is of high commercial value.In West Africa, the seeds of P. biglobosa provide a rich source of vegetable protein for human food and livestock feed.They are usually fermented to a tasty condiment used as a flavour intensifier for soups and stews (Dike and Odunfa, 2003).The husks and pods are good feed for livestock (Obizoba, 1998).Fetuga et al. (1974) reported the plant to be a good source of tannins, saponins, gums, fuel and wood.The seeds of various species of the genus Parkia have been investigated for their protein and amino acid contents.However, the use of African locust bean seeds as protein source is limited by the presence of inherent *Corresponding author.E-mail: toajibade@gmail.com.Tel: +234-7032936615.
anti-nutritional factors that cause poor protein digestibility in man and animals and are capable of precipitating other deleterious effects (Osagie, 1998;Soetan and Oyewole, 2009).
Various workers have shown that haematological investigations could be used to evaluate the physiological status of an animal (Kakade et al., 1974;de Gruchy, 1976) and an assessment of haematological parameters can be used to determine the extent of deleterious effect of foreign compounds including plant extracts on the blood.Also, Cells naturally contain enzymes for their functions and damages to cellular membrane normally leads to the escape of the enzymes into the blood where their presence or activities can easily be measured as an index of cellular integrity (Coles, 1989;Coppo et al., 2002).Further, certain plasma biochemistry could be used to identify tissue damage (Patti and Kulkarni, 1993).For instance, aspartate aminotransferase (AST), alanine amino-transferase (ALT) and alkaline phosphatase (ALP) were normally found within the cells of the liver, heart, kidneys, muscles and organs.Their increase concen- tration in the plasma indicate tissue injury or organ dysfunction (Wells et al., 1986).
Although P. biglobosa is used extensively in West Africa for food and medicinal purposes, the effect of the seed or its extract on haematology and plasma biochemistry are not well documented.Therefore, the aim of this study was to evaluate the effects of aqueous extract of P. biglobosa seeds on haematological and plasma biochemical parameters in rats in order to confirm its safety or otherwise on these parameters which are useful in evaluating the physiological state of the animal.

Preparation of plant extract
The seeds of P. biglobosa obtained from the Botanical Garden, University of Ibadan, were air dried, pulverized and subjected to cold aqueous extraction.The extraction was carried out as described by the method of Njar et al. (1993) and Raji (1995).The pulverized seeds weighing 1000 g poured into a muslin bag were soaked in 4.5 L of distilled water, for 72 h.The extract was obtained after filtration of the supernatant with Whatman No.1 filter paper.Subsequently, complete removal of the solvent from the supernatant, was achieved with a water bath placed in a fume cupboard because of the pungent smell emanating from it.A sticky, dark brown coloured extract weighing 154.61 g (a yield of 15.46%) was obtained after boiling on the water bath for about 12 h.The resulting extract was reconstituted in distilled water to give final concentrations of 100, 200 and 400 mg/ml by dissolving 10, 20 and 40 g of the extract in 100 ml of distilled water, respectively.

Experimental animals
Twenty eight healthy male white Wistar strain albino rats (200 to 240 g) of both sexes, obtained from the Animal House, Faculty of Veterinary Medicine, University of Ibadan, were used for the study.The rats were fed with rat cubes (Ladokun Feeds Limited, Ibadan, Nigeria) and water ad libitum, acclimated to laboratory conditions for one week, and randomly divided into four groups (control, groups 1, 2 and 3).A varied dosage of the seed extract at 200, 400 and 800 mg/kg, dissolved in distilled water was administered orally, by means of bulbed steel needle to rats in groups 1, 2 and 3, respectively.The rats in the control group were administered distilled water, orally.Rat was chosen as the experimental animal for the study because toxic substances readily produce demonstrable effects in rats (Farris and Griffith, 1949).

Haematology and plasma biochemical studies
At the end of the 21 days of treatment, each rat was bled through the orbital sinus into heparinised bottles for haematological studies.Packed cell volume (PCV) and haemoglobin concentration were determined by the microhematocrit and cyanmethaemoglobin methods, respectively as described by Jain (1986).Erythrocyte count was determined by the haematocytometry method as described by Jain (1986).Total white blood cell (WBC) count was made in a haemocytometer using the WBC diluting fluid and differential leucocytes counts were made by counting the different types of WBC from Giemsa stained slides viewed from each of the 30 fields of oil immersion objective of a microscope (Coles, 1989).Erythrocyte indices including mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined from the values obtained from red blood cells (RBC) count, haemoglobin concentration and PCV values (Duncan et al., 1994).
From the plasma, total protein was measured using biuret reaction, while albumin was measured by colorimetric estimation using sigma diagnostic reagent (Sigma Diagnostic, UK.), which contained bromocresol green (BCG).Globulin was obtained from difference of total protein and albumin.Aspartate and alanine aminotransferases were determined using a photoelectric colorimeter as described by Duncan et al. (1994).Blood urea nitrogen and creatinine levels were also determined using photoelectric colorimeter as described by Coles (1989).Gammaglutamyl transferase (GGT) was measured using the kinetic photometric method (Szaszi, 1969).

Statistical analysis
All values were expressed as mean ± standard error of mean (S.E.M).The means and standard error of means as well as oneway analysis of variance (ANOVA) for statistical significance was determined using GraphPad Prism, version 5.0, San Diego, CA.P values at 5% were regarded as significant.

RESULTS
The results of the effects of the aqueous extract of P. biglobosa seeds on haematological parameters are presented in Tables 1 and 2. Changes induced by the extract on erythrocyte values of treatment groups were not significantly different (p>0.05) from the control, except for increases in red blood cell count and packed cell volume, which are significant (p<0.05) at 800 mgkg -1 dose and haemoglobin, which is significant (p<0.05) at  400 and 800 mgkg -1 dose.Also, the extract induced significant (p<0.05)increase in total white blood cell count and neutrophil at 800 mgkg -1 , and 400 and 800 mgkg -1 dose caused significant (p<0.05)increase in lymphocyte values.Changes produced in eosinophil and monocyte values of the treatment groups are not significantly different from the control.

Effects of extract on plasma biochemistry of rats
The result of the effects of the aqueous extract of the seeds of P. biglobosa on plasma biochemical values is presented in Table 3.The extract caused significant (P<0.05)increase in total protein and albumin at the 400 and 800 mgkg -1 dose.Aspartate amino transferase (AST) also significantly increased at the 800 mgkg -1 dose.The extract, at the 800 mgkg -1 dose caused signi-ficant decrease in the level of blood urea nitrogen (BUN), and at 400 and 800 mgkg -1 dose caused significant decrease in the level of creatinine.However, changes observed in plasma levels of globulin, alanine amino transferase (ALT) and GGT were not significantly (P>0.05)different from the control at all treatment doses.

DISCUSSION
The main function of red blood cells is the transportation of oxygen into tissues of the body.Any pathological condition that affects the red blood cell alters its function and this may be detrimental to the body (Agbor et al., 2005).In this study, there were no obvious haemolytic changes on red blood cells, haemoglobin, packed cell volume, mean corpus-cular volume and mean corpuscular haemoglobin concentration of rats treated with the P. biglobosa seed extract.Rather, the extract significantly increased some of these parameters.The absence of significant decrease on the erythrocyte indices suggests that the extract does not possess toxic substances that can cause anaemia and the induction of significant increases in erythrocyte count, packed cell volume and haemoglobin strongly suggests that P. biglobosa seed extracts have haematinic or haematopoeitic properties.Substances that demonstrate significant effect on these parameters would have effects on bone marrow, kidney and also haemoglobin metabolism (Young and Maciejewski, 1997).Plant extracts with oxygen scavenging activity may compete with haemoglobin in erythrocytes for oxygen resulting in hypoxia which then stimulates haemoglobin synthesis and erythrocytes production (Song et al., 1987).It is also possible that the end product of meta-bolism of the seed extract, in vivo, stimulates the kidney directly to cause formation and secretion of erythrogenin which converts erythropoietinogen to erythropoietin, a potent stimulator of the bone marrow.
Estimated total number of white blood cells in the blood varies according to age, sex and more importantly, the physiological conditions of the body.Reduction in white blood cell, neutrophil and lymphocyte counts is positively correlated with susceptibility to infection, leukaemia and possible compromise of cellular and humoral mediated immunity (Bochner et al., 1991).In this study, the extract of the seeds of P. biglobosa caused significant increase in total white blood cell count, neutrophil and lymphocyte values at 800 mgkg -1 dose.Therefore, it can be inferred that the extract has immunoprotective effect because white blood cells are involved in the cellular and humoral defense mechanisms of the body and are responsible for fighting against foreign agents.The significant rise in lymphocytes also points to the potential usefulness of this plant as an immune system stimulating agent (Kinney et al., 1999).
Estimation of plasma albumin is a good criterion for assessing the function and secretory capacity of the liver (Yakubu et al., 2005) because albumin is the protein with the highest concentration in plasma and it transports many small molecules in the blood (for example, bilirubin, calcium, progesterone and drugs).It also prevents the fluid in the blood from leaking out into the tissues (Duncan et al., 1994).The significant decrease of extract of P. biglobosa on the levels of albumin at 200 and 800 mgkg -1 and total protein at 400 and 800 mgkg -1 may suggest that the extract has ability to inhibit in vivo protein biosynthesis.However, the extract caused significant increase in total protein at 400 mgkg -1 .Therefore, the dose is an important factor.Alanine and aspartate transferases (ALT and AST) are well known transaminases used as biomarkers to predict possible toxicity in the blood of sick animals (Akdogan et al., 2003).These enzymes are only released into the blood in significant amounts from the cytosol and subcellular organelles when hepatic injuries occur (Lu, 1996).It is known that an increase in concentration of ALT, AST and alkaline phosphatase in the serum directly reflects a major permeability, congestion or cell rupture (Pieme et al., 2006;Tedong et al., 2008).However, ALT is more hepatospecific than AST because it is more sensitive to hepatic damage (Herfindal and Gourley, 2000).Elevations in ALT levels are rarely observed except in chronic liver disease (Gad, 2001).Therefore, the lack of significant effect of the extract on ALT implies that oral administration of the extract did not cause serious impairment to liver function.The non-significant effect on GGT further attests to this fact.It is however possible that the extract could have deleterious effects on other organs at high doses, or when used for longer duration.

Conclusion
Aqueous extract of the seeds of P. biglobosa does not contain deleterious chemical substances capable of altering haematological values or cause blood dys-Ajibade and Soetan 15449 functions in rats.The seeds may be helpful as immunomodulating agent because of the effects observed on white blood cells.These findings justify the use of the seeds of P. biglobosa for nutritional and medicinal purposes.

Table 1 .
Effects of aqueous extract of P. biglobosa seeds on erythrocyte values.

Table 2 .
Effects of aqueous extract of P. biglobosa seeds on leukocyte values.

Table 3 .
Effect of aqueous extract of P. biglobosa seeds on plasma chemistry.