Liver and Kidney Function Tests and Histological Study on Malaria Parasite Infected Mice Administered with Seed Extract of Picralima nitida

The liver and kidney are very important organs of the body that play vital roles of detoxification of compounds and removal of waste products respectively. In this research, some liver and kidney function tests were carried out on albino mice infected with malaria parasite to know the effect of treatment with extract on them. Pulverised dried seeds of Picralima nitida were extracted using ethanol. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, total bilirubin, total protein, urea and creatinine levels and histopathological studies on liver and kidney tissues were performed. From the results of the experiment, significant (p < 0.05) reductions were observed in serum levels of urea, creatinine of the treated groups compared to the positive control (group infected with malaria parasite and treated with 3% tween 80) on days 3 and 5 post treatment. There was non-significant (p > 0.05) reduction in serum AST and ALT activities of the groups treated with 20, 40 and 80 mg/kg b.w. of the extract on days 3 and 5 post treatment compared to the positive control. Groups treated with 20 and 40 mg/kg b.w. of the extract showed significant (p < 0.05) reduction in bilirubin level on days 3 and 5 post treatment compared to the positive control. All the treated groups showed significant (p < 0.05) elevation in serum total protein on day 3 post treatment while the group treated with 80 mg/kg b.w. of the extract showed significant (p < 0.05) elevation in total protein on day 5 treatment compared to the positive control. Histopathological studies on the hepatic tissues of the treated mice showed lesser inflammatory cells around the portal areas than the untreated. No lesions were noticed in the renal tubules of both the treated and untreated mice. From the results of this experiment, it is now obvious that Picralima nitida seeds possess good hepato-protective properties.


INTRODUCTION
Plasmodium berghei is a unicellular protozoan parasite that infects mammals other than humans. It was first described by Vincke and Lips in 1948 in the Belgian Congo. It is found in the forests of Central Africa, where its natural cyclic hosts are the thicket rat and the mosquito (Anopheles dureni) [1].
Like all malaria parasites of mammals, P. berghei infects the liver after being injected into the bloodstream by a bite of an infected female mosquito. After a short period of development and multiplication, these parasites leave the liver and invade the erythrocytes. P. berghei infections may also affect the brain and can cause cerebral complications in laboratory mice. The complete genome of P. berghei has been sequenced and it shows a high similarity both in structure and gene content with the genome of the human Plasmodium falciparium [2,3].
The parasite can be genetically manipulated and so, is often used for the analysis of the function of malaria genes using the technology of genetic modification [4,5]. A number of genetically modified Plasmodium berghei cell lines have been generated and these transgenic parasites are tools to study and visualize the parasites in the living host [6,7].
Livers function tests (LFTs of LFs) are group of clinical biochemistry laboratory blood assays designed to give information about the state of a patient's liver [8]. The parameters measured include prothrombin time (PT/INR), activated partial thromboplastin time (a PTT), albumin, bilirubin and others. Aspartate aminotransferase and alanine aminotransferase (AST and ALT) are useful biomarkers of liver injury in a patient with some degree of intact liver [9]. Some tests are associated with functionality (e.g. albumin), some with cellular integrity (e.g. transferases), and some with conditions linked to the biliary tract (e.g. gamma-glutamyl transferase and alkaline phosphate) [10]. Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver diseases, distinguish among different types of liver disorders, guage the extent of known liver damage and follow the response to treatment.
Albumin is the main constituent of total protein made specifically by the liver and can easily be measured. Its levels are decreased in chronic liver disease, such as cirrhosis. It is also decreased in nephritic syndrome, where it is lost through the urine. The consequence of low albumin can be edema since the intravascular oncotic pressure becomes lower than the extravascular space [11].
Alanine aminotransferase (ALT), also known as serum glutamate pyruvic transaminase (SGPT), is a member of transferase family. It catalyses the reversible reaction in which an α-amino group is transferred between alanine and glutamine [12].
This enzyme is found mainly in liver and small amount in heart, muscle, and kidney. In healthy subjects, serum ALT levels are low. However, when cells are damaged, for instance, acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction, this enzyme may leak into the blood stream and its levels are significantly elevated [13,14].
Aspartate aminotransferase (AST) is similar to ALT in that it is another enzyme associated with parenchymal cells. It is raised in acute liver damage, but is also present in red blood cells, and cardiac and skeletal muscles, so is not specific to the liver [15]. So, elevated AST levels are not specific for liver damage. Aspartate aminotransferase, as with all aminotransferases, operates via dual substrate recognition; that is, it is able to recognize and selectively bind two amino acids (Asp and Glu) with different sidechains [16]. In either case, the aminotranferase reaction consists of two similar half-reactions that constitute what is referred to as a ping-pong mechanism. In the first half-reaction, amino acid 1 (e.g., L-Asp) reacts with the enzyme-PLP complex to generate ketoacid 1 (oxaloacetate) and the modified enzyme-PMP. In the second half-reaction, ketoacid 2 (α-ketoglutarate) reacts with enzyme-PMP to produce amino acid 2 (L-Glu), regenerating the original enzyme-PLP in the process.
Total bilirubin includes both unconjugated and conjugated bilirubin. Unconjugated bilirubin is a breakdown product of haem [17]. It is very hydrophobic and is mainly transported bound to albumin circulating in the blood. High concentrations of hydrophobic drugs and high free fatty acids can cause elevated unconjugated bilirubin. The liver is responsible for clearing the blood of unconjugated bilirubin, and about 30% of it is taken up by a normal liver on each pass of the blood through the liver by the some mechanisms.
Bilirubin can be conjugated with a molecule of glucuronic acid which makes it soluble in water. Some of the double-bonds in bilirubin isomerizes when exposed to light. This is used in the phototherapy of jaundiced newborns: the E, Zisomers of bilirubin formed upon light exposure are more soluble than the unilluminated Z, Zisomer, as the possibility of intramolecular hydrogen bonding is removed [18]. This allows the excretion of unconjugated bilirubin in bile.
Erythrocytes generated in the bone marrow are disposed of in the spleen when they get old or damaged. This releases haemoglobin, which is broken down to haem as the globin parts are turned into amino acids [19]. The haem is then converted into unconjugated bilirubin in the reticuloendothelial cells of the spleen. This waterinsoluble unconjugated bilirubin is then bound to albumin and sent to the liver. In the liver, bilirubin is conjugated with glucuronic acid by the enzyme glucuronyltransferase making it water-soluble and afterwards goes into the bile. Conjugated bilirubin is not absorbed in the small intestine but instead is passed into the colon [19]. Colonic bacteria deconjugate and metabolize the bilirubin into colourless urobilinogen which is further oxidized to urobilin and stercobilin which give stool its characteristic brown colour. A trace amount of urobilinogen is reabsorbed into the enterohepatic circulation to be re-excreted in the bile: some of this is instead processed by the kidneys colouring the urine [20].
Kidney or renal function tests are common laboratory tests used to evaluate how well the kidneys are working. Such tests include: blood urea nitrogen (BUN), serum creatinine test, urinalysis and glomerular filtration rate (GFR) test [21]. Urea is produced as a waste product of protein digestion by the liver. Normal human adult blood should contain 6-20 mg/dL of blood. The main causes of an increase in BUN are: high protein diet, decrease in glomerular filtrate rate (GFR) and in blood volume, congestive heart failure, gastrointestinal haemorrhage, fever and increased catabolism [22]. Decreases in BUN are caused by severe liver disease, anabolic state and syndrome of inappropriate antidiuretic hormone (ADH).
Creatinine is a breakdown product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body [23]. Serum creatinine is an important indicator of renal health because it is an easily measured by-product of muscle metabolism excreted unchanged by the kidneys. It is synthesized by the liver from the methylation of glycocyamine by S-adenosyl methionine. It is then transported through the blood to the other organs, muscles, and brain where it is phosphorylated to phosphocreatine. During this reaction, creatine and phosphocreatine are catalyzed by creatine kinase, and a spontaneous conversion to creatinine may occur [24]. Creatinine is removed from the blood by the kidneys through glomerular filtration and also by proximal tubular secretion [25]. There is little or no tubular reabsorption of creatinine. When its filtration in the kidney is impaired, blood creatinine levels rise and so its levels in blood and urine may be used to calculate the creatinine clearance (Crcl). Creatinine levels in the blood and urine correlates with the glomerular filtration rate (GFR) while blood creatinine levels alone are used to calculate the estimated GFR (eGFR).
Extracts of Picralima nitida parts (root, seed and stembark) have been shown to exhibit a broad spectrum of activity against bacterial strains [26]. Among the susceptible bacteria were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Salmonella kintambo. The Minimum Inhibitory Concentration (MIC) values for the ethanol extracts range from 6.25 to 50 mg/ml, while the MIC values for the cold water seed extracts (CWs) was 50 mg/ml showing that the ethanol extracts were more potent than the cold water extracts at very low concentrations. The activities of these extracts were possible because they contained a good number of phytochemicals such as alkaloids, saponins, flavonoids, steroids, tannins and terpenoids. Ubulom et al. [27] investigated into the antifungal activity of both aqueous and ethanolic extracts of Picralima nitida seeds against Aspergillus flavus, Candida albicans and Microsporum canis and found them to have activity against these organisms. However, the ethanol extract was found to have better antifungal activity than the aqueous extract. Okonta and Aguwa [28] compared the hypoglycemic effect of glycosides extract with that of alkaloids extract of P. nitida seed and were able to establish that the hypoglycemic effect of the seed lies in the glycosides extract. The alkaloids extract reduced given i.p caused increase in mean fasting blood glucose levels while the glycosides extract reduced the blood glucose levels in normoglycemic and hyperglycemic rats.
In traditional medicine, numerous plants have been used for cognitive disorders, including memory loss and antiaging. In accordance, Picralima nitida fruits and seeds have been found to possess memory enhancing property [29] and so could be applied in the treatment of cognitive disorders like Alzheimer's disease, amnesia, depression and schizophrenia. Soladoye et al.
[30] performed ethnobotanical survey of plants used in the treatment of haemorrhoids in South-Western Nigeria and found Picralima nitida fruit to be very effective. From the survey, a total of 143 plant species belonging to 58 families were found to be useful for the treatment of haemorrhoids. The most prominent family among these plant families is the Leguminosae, and then others include Euphorbiaceae, Apocynaceae, Meliaceae, Compositae and Poaceae. The seed extract of P. nitida [31] showed inhibition of β-glucosidase activity and so the exploitation of this potential could be of immense value in the current search for new therapeutically effective drugs with inhibitory effects against β-glucosidase which is implicated in HIV infectivity of cells. The aim of this study however, is to ascertain the protective effect of the seed extract of Picralima nitida on hepatic and renal tissues so as to advice the users better on how to administer the drug.

Collection of Picralima nitida seeds
The seeds of Picralima nitida were collected from Isuofia, Aguata Local Government Area of Anambra State, Nigeria and were authenticated by Mr. Alfred Ozioko of the Bioresource Development and Conservation Programme (BDCP) Research Centre, Nsukka, Nigeria.

Chemicals/reagents
The commercial kits used for this study were products of Randox, QCA, USA and Biosystem Reagents and Instruments, Spain.

Extraction
Seeds of Picralima nitida plant were harvested and then dried at room temperature (29-35 o C) for three weeks, after which they were pulverized into powdered form with a Creston high speed milling machine. The powdered seeds (1 kg) were then macerated in 5 volume (w/v) absolute ethanol at room temperature for 48 hours. Afterwards, the extract was filtered through muslin cloth on a plug of glass wool in a glass column. The resulting ethanol extract was finely filtered using Whatman qualitative filter paper, concentrated and evaporated to dryness using rotary evaporated at an optimum temperature of between 40 and 45ºC (to avoid denaturation of the active ingredients). The concentrated extract was stored in the refrigerator for subsequent studies.

Experimental design
A total of 180 albino mice of either sex weighing 20-34 g were housed in separate cages, acclimatized for one week and then divided into six groups of thirty mice each as follows: The route of administration (treatment) was via oral route with the aid of an oral intubation tube.

Procurement of parasitaemia
Malaria parasitaemia (Plasmodium berghei) was obtained from malaria-infected mice at Veterinary Medicine Department of University of Nigeria, Nsukka. Ten drops of the parasitized blood obtained with the aid of a capillary tube through the ocular region of the mice, was diluted with 1 ml of normal saline. Thereafter, 0.2 ml of the diluted parasitized blood was used to passage three mice that served as the host from where subsequent ones were passaged.

Assay of aspartate aminotransferase (AST) activity
The activity of aspartate aminotransferase (AST) was assayed by the method of Reitman and Frankel [32] as outline in Randox kit.
Aspartate aminotransferase activity was measured by monitoring the formation of oxaloacetate hydrazone with 2, 4dinitrophenylhydrazine.
The AST substrate phosphate buffer solutions (0.5 ml each) were pipetted into both the reagent blank and sample test tubes. Serum sample of 0.1 ml was added to the sample test tube only and mixed thoroughly. Then, 0.1 ml of distilled water was added to the reagent blank. Afterwards, the entire reaction medium was well mixed and incubated in a water bath at 37 0 C for 30 minutes.
A volume, 0.5 ml of 2, 4-dinitrophenylhydrazine was added to the reagent blank and the sample test tubes immediately after incubation. Also, 0.1 ml of the sample was added to blank only. The medium was mixed and allowed to stand for exactly 20 minutes at 25ºC. Finally, 5.0 ml of sodium hydroxide (NaOH) solution was added to both the blank and sample test tubes and mixed thoroughly. After 5 minutes, the absorbance of sample was read at 550 nm against the sample blank after 5 minutes. The activity of AST in mice serum was obtained from the already calibrated table.

Assay of alanine aminotransferase (ALT) activity
The activity of alanine aminotransferase was assayed by the method of Reitman and Frankel [32] as outlined in the Randox kit.
Alanine aminotransferase is measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4dinitrophenylhydrazine.
Aliquots, 0.5 ml of the ALT substrate phosphate buffer solutions were pipetted into two sets of test tubes labeled B (sample blank) and T (sample test). The serum sample (0.1 ml) was added to the sample test (T) test tubes only and mixed thoroughly and then, incubated in a water bath at temperature of 37ºC for 30 minutes.
A volume, 0.5 ml of 2, 4-dinitrophenylhydrazine was added to each of the test tubes labelled T (sample test) and B (sample blank) immediately after the incubation. Also, 0.1 ml of serum sample was added to sample blank (B) only. The entire medium was mixed thoroughly and allowed to stand at 25ºC for exactly 20 minutes. After this, 5.0 ml of sodium hydroxide (NaOH) solution was added to each test tube and mixed thoroughly. The absorbance of sample against the sample blank was read at a wavelength of 550 nm against the sample blank after 5 minutes.
The activity of ALT in the serum was obtained from a pre-calibrated table.

Determination of total bilirubin concentration
Total bilirubin concentration was determined using the method of Jendrassik and Grof [33] as outlined in the Randox kit.
A volume, 0.2 ml of sulphanilic acid was pipetted into the sample blank tube and sample tube. This was immediately followed by the addition of 0.05 ml of sodium nitrite to the sample tube. Caffeine (10 ml) and 0.2 ml of sample were also pipetted into each of the sample blank tube and sample tube. These mixtures were mixed and incubated for 10 minutes at 20-25ºC. Finally, 1.0 ml of tartrate was pipetted into the sample blank tube and sample tube. These mixtures were once again mixed, incubated at 20-25ºC for 30 minutes and their absorbances read at 578 nm against the sample blank.

Determination of Total Protein
Total protein concentration was determined using the method of Lowry et al. [34].
Eleven sets of test tubes were set up in a rack and filled with different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µl) of Bovine Serum Albumin (BSA). A known volume, 2 ml of solution D (48 ml of 2 % Na 2 CO 3 in 0.1 N NaOH; 1 ml of 1 % NaK Tartarate in H 2 O and 1 ml of 0.5% CuSO 4 .5H 2 O in H 2 O). The mixture was incubated for 10 minutes at room temperature. An aliquot, 0.2 ml of dilute Folin-phenol as added to each test tube and vortex immediately. Afterwards, the contents were incubated for 30 minutes and the absorbance readings taken at 600 nm.

Determination of serum urea concentration
The concentration of serum urea was determined using the method of Tietz [35] as outlined in Randox kits, UK.
A known volume, 10 µl of the sample was pipetted into the sample tube; 10 µl of the standard was also pipetted into the standard tube while 10 µl of distilled water was added to the blank tube. A volume, 10 µl sodium nitroprusside and 10 µl urease were added to each of the three tubes. The tubes were mixed and incubated at 37ºC for 15 minutes. Then, 2.50 ml of phenol was added to each of the three tubes followed by addition of 2.50 ml of sodium hypochlorite. After mixing, they were incubated at 37ºC for 15 minutes and the absorbance of the sample was read against the reagent blank at 546 nm.

Determination of serum creatinine concentration
The concentration of serum creatinine was determined using the method of Tietz [35] as outlined in Randox kits, UK.
A volume, 100 µl, of distilled water was pipetted into the blank tube. Also 100 µl of the standard was added to the standard tube while 100 µl of the sample was pipetted into the sample tube. Then, 100 µl of the working reagent was pipetted into each of the three tubes and mixed. The absorbance of the sample was read against the blank at 492 nm.

Histopathological Study
The tissue specimens (liver and kidney) were excised from the sacrificed rats and fixed in 10% formaldehyde buffered saline (formal saline). After some days, the processing started with the dehydration in graded levels of alcohol (about 70-100%) in ascending order to remove the water content.
After the dehydration, the tissues were cleared in xylene impregnated with paraffin wax and sectioned at 5 microns thickness using rotary microtone. The sections were floated on a water bath maintained at a temperature of 2-3ºC below melting point of the paraffin wax after which the sections were dried on a hot plate maintained at a temperature of 2-3ºC above the melting point of the paraffin wax used. After proper drying, the sections were stained and mounted using haematoxylin and eosin in such a way as to avoid air bubbles.

Experimental Animals
The animals used for this study were white albino mice of either sex weighing 20-34 g. The mice were between 3-4 months old and were obtained from the Animal Unit of Faculty of Veterinary Medicine, University of Nigeria, Nsukka.
Clearance and approval for the humane use and handling of laboratory animals were given by the ethical committee of Biochemistry Department, University of Nigeria Nsukka.

RESULTS
In Table 1, the serum AST and ALT activities and the total protein level are presented. In normal mice, the AST activity of found to be 28.60±1.17 IU/L; the ALT activity was 44.60±2.78 IU/L and the total protein level was 6.74±0.19 g/dl. In the positive control group, AST and ALT activities was non-significantly (p > 0.05) elevated compared to the normal control. The total protein level in the positive control was significantly (p < 0.05) reduced compared to the normal control. In In Table 2  The histomorphological changes in the liver sections of the different groups of mice (positive/untreated control, negative/normal control, standard control and treated groups) are shown in Plates 1. There is infiltration of mononuclear cells (inflammatory cells) around the portal area (PA) (portal hepatitis) (white arrow) in groups 2,5 and 6 while group 1 shows normal PA and hepatocytes (black arrow). The severity of inflammatory cells infiltration is more in 2 than 5 and 6.
The changes in the kidney sections of the different groups of mice (positive/untreated control, negative/normal control, standard control and treated groups) are presented in Plates 2. There is no infiltration of mononuclear cells around the glomeruli (G) and renal tubules (arrow) in the kidneys of all the experimental groups.

Plate 1. Photomicrograph of liver section of mice from experimental groups showing A-normal control, B-infected but untreated, C-infected and treated with artesunate, D-infected and treated with extract. The mononuclear infitration of cells around the portal area-portal hepatitis (white arrow) in groups B,C and D while the group A shows normal portal area(PA) and hepatocytes(black arrow). Note that the severity of inflammatory cells infiltration is more in B than D and C. H and E x 400.
A B D C [37] who asserted that serum ALT and AST activities were significantly higher in positive control (group inoculated with malaria but not treated) when compared with group treated with graded doses of Aframomum sceptrum.
Urea [22] is produced as a waste product of protein digestion by the liver which is normally removed from the body through the process of utrafiltration in the glomeruli of the kidneys.  1.14 ± 0.01* 1.07 ± 0.01 " 5 1.14 ± 0.02* 0.99 ± 0.02 " 6 1.07 ± 0.02 1.03 ± 0.02 " which stated that extracts of Carica papaya, Alstonia boonei and Clerodendrum violaceum Gürke have been observed to play vital roles in decreasing the serum urea and creatinine concentrations thus proving the efficacy of extracts in maintaining the integrity of the kidney function.
A rise in serum bilirubin concentration is an indication of liver damage, parenchyl or biliary [40]. Bilirubin was found to be produced in highest concentrations by group 3 and in least amounts by group 5 on days 3 and 5 of post treatment. The standard control showed better bilirubin clearance effect on mice than the extract treated groups on days 3 and 5 of post treatment. Only group 5 showed a significant decrease in serum total bilirubin concentration among the extract treated groups when compared with the positive control indicating that this extract has bilirubin clearance effect at higher doses. The evidence showing that extract treatment caused a dose-dependent decrease in total bilirubin concentration revealed that this extract enhance the liver function of clearing the blood unconjugated hydrophobic bilirubin by combining it with glucuronic acid making it watersoluble conjugated bilirubin using the enzyme UDP-glucuronide transferase. The time dependence of the effect of the treatment (Tables 1) showed greater effect on day 5 than day 3 of post treatment. This correlated with observation of Olorunnisola and Afolayan [41] who showed that leaf extract of Sphenocentrum jollyanum administered to malaria-infected mice significantly decreased the total bilirubin concentration. In conformation to the present study too, crude aqueous extract of the fruiting body of medicinal mushroom, Ganoderma lucidum has been found to significantly reduce the total bilirubin level in malaria-infected mice [42].
Decreased levels of albumin, the main constituent of total protein are noticed in chronic liver diseases such as cirrhosis and nephritic syndrome where it is lost through the urine [11]. The highest protein concentrations were produced by group 3, followed by group 4 and the least by group 3 on days 3 and 5 of post treatment. The extract treated groups produced better total protein that the standard control on days 3 and 5 of post treatment. The evidence showing that the extract treatment caused a dose-dependent increase in total protein revealed that the extract protected integrity of the liver which is involved in manufacturing of total proteins. The time dependence of the effect of the treatment (Table 1) showed greater effect on day 5 than day 3 of post treatment.
The histomorphological changes in the liver sections of the different groups of mice (Plates 1 -4) showed inflammatory cells around the portal area (portal hepatitis) in groups 2,5 and 6 while group 1 shows normal portal area (PA) and hepatocytes. The severity of cellular infiltration (inflammation) is more in 2 than 5 and 6; an indication that the extract produced hepatoprotective effect on the liver of the treated mice. The histomorphological changes in the Kidney sections of the different groups of mice (Plates 5-8) showed normal glomeruli(G) and renal tubules in the kidneys of all the experimental groups.

CONCLUSION
The decreased activities of the liver function enzyme markers, serum bilirubin level and increase in total protein of the treated groups are indicatives that the extract has hepatoprotetective effect. The functionality of the kidney was enhance by the administration of this extract thus serum amounts of urea and creatinine were significantly reduced in the treated mice. Nevertheless, prolonged treatment and higher doses with such extract can pose a seroius danger and damage to these organs. The observed normal glomeruli and renal tubules in the untreated and treated groups shows that the kidney is not a target organ for the malaria parasite during its exo-erythrocytic stage and also indicates the apparent less toxic effect of the extract on body tissue.