Research Article Effect of Aqueous Stem Bark Extract of ...downloads.hindawi.com/journals/jt/2013/803835.pdfKhaya senegalensis for twenty-eight days at the experimental dose resulted

Hindawi Publishing CorporationJournal of ToxicologyVolume 2013 Article ID 803835 9 pageshttpdxdoiorg1011552013803835

Research ArticleEffect of Aqueous Stem Bark Extract ofKhaya senegalensis on Some Biochemical Haematologicaland Histopathological Parameters of Rats

A Onu1 Y Saidu1 M J Ladan1 L S Bilbis1 A A Aliero2 and S M Sahabi3

1 Department of Biochemistry Usmanu Danfodiyo University PMB 2346 Sokoto Nigeria2 Department of Biological Sciences Usmanu Danfodiyo University PMB 2346 Sokoto Nigeria3 Department of Histopathology Usmanu Danfodiyo University PMB 2346 Sokoto Nigeria

Correspondence should be addressed to A Onu andrewonuyahoocom

Received 12 July 2013 Accepted 19 September 2013

Academic Editor Abdelwahab Omri

Copyright copy 2013 A Onu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The subchronic effect of aqueous stem bark extract ofKhaya senegalensis on some biochemical haematological and histopathologi-cal parameters of rats was investigatedThe rats were divided into six groups of five rats per group Groups I to VI were administeredgraded doses of 0 400 800 1200 1600 and 2000mgkg bw respectively The result of study revealed that administration of theKhaya senegalensis for twenty-eight days at the experimental dose resulted in significant (119875 lt 005) increase in urea electrolytes(Na+ K+) and creatinine levels The extract also significantly (119875 lt 005) increased serum activity of ALT AST and ALP Thelevels of protein albumin and bilirubin were significantly changed when compared to their control values but they were not dosedependentThe hematological indices assayed in this study were not significantly affected at the experimental dose when comparedto the control values Histological studies of the liver showed cellular degeneration and necrosis and bile duct hyperplasia andfibrosis with lymphocytic infiltration of the hepatocyte providing supportive evidence for discussing the biochemical findingsindicative of functional derangementThe histological architecture of the kidney and that of the heart were however preservedTheresult of this study indicates that the aqueous stem bark extract of K senegalensismay affect the cellular integrity of vital organs ofthe body

1 Introduction

The therapeutic value of medicinal plants has long beenexploited for the management of various disease conditionsin traditional practice This practice has gained appreciableacceptance in health care delivery in developing and devel-oped nations with the notion that they are relatively harmlessbut research is beginning to show that some of them may betoxic

Khaya senegalensis (Desr) A Juss (Family Meliaceae) isa tree that is widely distributed in the sub-Saharan savannahfrom Senegal to Sudan and Uganda It is a round evergreencrown of dark shiny foliage pinnate leaves and characteristicround capsules that grows up to 40m high [1] The thera-peutic value of Khaya senegalensis has been recognized indifferent systems of traditional medicine for the treatment of

various conditions The decoction of the stem bark extract iscommonly used for treating jaundice dermatoses malariafever mucous diarrhea and venereal diseases as well as forhookworm infection and a taeniacide remedy [2 3] Khayasenegalensis extracts have been reported to exhibit anti-inflammatory effects [4] as well as anti-bacterial [5] anti-helmintic [6] antitumor antioxidant [7] and antiplasmodialactivities [8] The stem bark extract and the chemical con-stituent profile have been the subject of extensive phytochem-ical and pharmacological investigations since the 1960s [9ndash11] Despite the extensive research on the stem bark extractof Khaya senegalensis a dearth of information on the toxico-logical profile of this plant still exists This work is thereforedesigned to evaluate the acute and subchronic toxicity effectsof the extract on the liver and kidney

2 Journal of Toxicology

2 Materials and Methods

21 Plant Materials Stem bark of K senegalensis was col-lected from the main campus of Usmanu Danfodiyo Univer-sity Sokoto (130 211015840 1610158401015840 N and 50 51015840 3710158401015840 E) in the month ofJune 2011The plant was identified by a taxonomistMal AMUmar from the Botany Unit Department of Biological Sci-ences Usmanu Danfodiyo University Sokoto and a voucherspecimen (UDUSVS201121) was prepared and deposited inthe herbarium of the same department

22 Experimental Animals Albino male rats weighing 150ndash200 g were purchased from the Animal House UsmanuDanfodiyo University Sokoto Nigeria They were housed inmetal cages and allowed free access to a standard laboratorypellet diet (ECWA Feed Nig Ltd) and water ad libitumunder a 12-hour light-dark cycle throughout the experimentalperiod

23 Preparation and Extraction of Plant Samples Plant mate-rials were washed and air-dried at room temperature untila constant weight was obtained The samples were groundusing a pestle and mortar and the powdered samples werethen bagged and stored in plastic bags until required

The plant sample was separately placed in an Erlenmeyerflask and one liter of distilled water was added into the flaskand it was allowed to soak for aminimumof 24 hours 5wvof the plant material was prepared The mixture was filteredusing a piece of clean sterile Muslin cloth to remove debrisand then filtered on Whatman filter paper The resultantfiltrate was evaporated to dryness [12] and the percentagerecovery was calculated (133)

24 Acute Toxicity Study Acute oral toxicity was performedusing the up- and down-procedure of the Organisation forEconomic Co-operation and Development [13] This proce-dure involves the limit and main test which was also usedfor the selection of a starting dose as well as determination ofLD50of the testing material

25 Subchronic Toxicity Study Animals were divided into sixgroups of five animals each The test substance was adminis-tered orally to the rats in groups IIndashIV in a gradation of 400800 1200 1600 and 2000mgkg body weight per day for 28days while group I (control group) animals were given anequivalent volume of normal saline orally for the same periodof 28 days under the same conditions

The body weight changes were monitored weeklythroughout the experimental period On the 29th day afteran overnight fast the rats were anaesthetized in a chloroformvapour and blood samples were collected from the animals bycardiac puncture into labeled Vacutainers for biochemicaland haematological assays Subsequently the animals weresacrificed by decapitation the organs (kidney liver andheart) were removed weighed and then fixed in 10 forma-lin for histopathological examinations [13]

26 Biochemical and Haematological Analysis Serum totalprotein was determined using the biuret method cupric ionsin the biuret reagent react with the peptide bonds of the pro-tein molecule in an alkaline solution to form a blue coloredcomplex [14] Albumin was determined using the Bromocre-sol Green method albumin binds with Bromocresol Greenin an acidic medium to form a blue-green complex whosecolour is proportional to the concentration of albumin in theserum [15] Transaminase activity (aspartate aminotrans-ferase (AST) and alanine aminotransferase (ALT))was deter-mined using the Reitman and Frankel [16] method ALT wasmeasured by monitoring the concentration of pyruvatedhydrazone that is formed with 24-dinitrophenylhydrazinewhile AST was measured by monitoring the concentrationof oxaloacetate hydrazone that is formed with 24-dinitro-phenylhydrazine Alkaline phosphatase (ALP) activity wasdetermined using the colorimetric method of Rec [17] thehydrolysis of 4-nitrophenyl phosphate at alkaline pH to4-nitrophenol and inorganic phosphate is catalysed by alka-line phosphatase and the intensity of the colour is directlyproportional to the activity of ALP Urea was investigatedusing the Urease-Berthelot colorimetric method of Fawcettand Scott [18] urea in serum is hydrolysed to ammonia in thepresence of urease The ammonia formed is measured spec-trophotometrically by the Berthelot reaction Creatinine wasmeasured by the picrate method [19] creatinine reacts withpicric acid in an alkaline medium to form a yellow-red col-ored complexwhich can bemeasured spectrophotometricallyat 520 nm Serum electrolytes Na+ and K+ were measuredusing a flame emission spectrophotometer Na+ and K+ areatomised using a flame the amount of atomisation is propor-tional to the quantity of the element in the solution whileHCO3was estimated using a titrimetric method [20] Packed

cell volume (PCV) of samples was determined using micro-hematocrit centrifuge at 12000 g for 5min [21] white bloodcell (WBC) count and differentials were determined bymechanical expansion and optical magnification augmentedby supravital cell staining [21]

27 Histopathological Analysis Three animals from eachgroup were selected randomly and dissected through a cen-tral abdominal incision The kidney heart and liver sampleswere collected and immediately fixed in 10 formal saline inlabeled sample plastic bottles The tissues were dehydrated ingraded concentrations of xylene embedded in molten paraf-fin wax and sectioned at 5 120583 Tissue sections were fixed onglass slides and stained with hematoxylin and eosin for lightmicroscopy at 400x [22] Photomicrographs of some of thetissues were taken using a microscope fitted with a cameraunit

28 Statistical Analysis Data were expressed as mean plusmnstandard deviation for the given number of observationsThe results were analyzed using one-way analysis of variance(ANOVA) followed by Dunnettrsquos multiple comparison testusing GraphPad Instat Software Differences were consideredsignificant when 119875 lt 005

Journal of Toxicology 3

CVB

K

S

H

H

N

NO

BD

(a)

S

N

L

(b)

Figure 1 Photomicrographs of liver sections fromcontrol dose group (a) and treated dose group (b)HampE stain 400x (a)Normal architectureof the liver showing the bile duct (BD) and central vein (CV) in the center of hepatic lobule filled with blood (B) Hepatocytes (H) arrangedin the form of cords are rounded in a polyhedral shape and radiate peripherally they show the nucleus (N) with clear nuclear membrane andnucleolus (No) and cords are separated by sinusoids (S) with Kupffer cells (k) (b) Normal architecture of the liver that show the nucleus (N)with a clear nuclear membrane and cords are separated by sinusoids (S) with Kupffer cells (k) with lymphocytes (L) surrounding the centralvein

Table 1 Effect of subchronic oral administration of aqueous stem bark extract ofK senegalensis on ratio of organ weight body weight of ratsand percentage change in body weight of the first day and last day of the experimental period

Dosemgkg bw

Kidney bodyweight Liver body weight Heart body weight Spleen body weight Change in body weight

()0 00053 plusmn 00001 00348 plusmn 00017 00028 plusmn 00005 00026 plusmn 00001 6264 plusmn 85

400 00057 plusmn 00003 00365 plusmn 00016 00029 plusmn 00008 00028 plusmn 00004 4711 plusmn 48b



1600 00057 plusmn 00003 00383 plusmn 000012b

00031 plusmn 00004 00034 plusmn 00005 3745 plusmn 48b

2000 00055 plusmn 00004 00398 plusmn 00017b

00031 plusmn 00005 00043 plusmn 00001b

3532 plusmn 99b

Values are expressed as mean plusmn SD 119899 = 5 The level of significance was set at b119875 lt 005 when compared with control at 0mgkg body weight Dunnettrsquos

multiple comparison test

3 Results

31 Acute Toxicity Study The LD50

was found to be greaterthan 5000mgkg body weight During the course of the toxi-city experiment the animals were observed for any change inbehavioural pattern physical characteristics and activityTherats in all the steps displayed no appreciable change in physi-cal or behavioural activities they fed well andmoved about intheir cages wellThe histological preparations of the liver aftertreatment with the extract are presented in Figures 1(a) and1(b) both the normal and experimental preparations appearwithin the range of a preserved histological architecture

32 Effect of Subchronic Oral Administration of Aqueous StemBark Extract of K senegalensis on Ratio of OrganWeight BodyWeight of Rats and Percentage Change in Body Weight of theFirst Day and LastDay of the Experimental Period Theeffects

of oral administration of aqueous extract ofKhaya senegalen-sis stem bark on the ratio of organ weight body weight ofexperimental rats and percentage change in body weight ofthe first and last days of the experimental period are shownin Table 1 Prolonged administration of the extract for 28 daysresulted in significant (119875 lt 005) decrease in percentagechange in body weight of the experimental rats in a dosedependent manner when compared to the values of thecontrol group Administration of several doses of the extractto their corresponding groups had no significant (119875 gt 005)effect on the ratio of kidney weight body weight as well asratio of heart weight body weight when compared to the val-ues of their corresponding control group However the ratiosof liver weight body weight and spleen weight body weightwere observed to be significantly different at the higher dosesof 1600mgkg (119875 lt 005) and 2000mgkg (119875 lt 001) whencompared to their corresponding control values


Table 2 Effect of subchronic oral administration of aqueous stem bark extracts of K senegalensis on kidney function parameters of rats

Dosemgkg bw Urea (mmolL) Creatinine Na+ (mmolL) K+ (mmolL)

0 383 plusmn 038 1447 plusmn 174 1343 plusmn 148 438 plusmn 008

400 487 plusmn 069b


800 492 plusmn 029b


1200 489 plusmn 037b


1600 507 plusmn 016b

4944 plusmn 942b

1408 plusmn 455b

558 plusmn 041b

2000 502 plusmn 020b

7046 plusmn 1099b

1428 plusmn 410b

621 plusmn 024b



Table 3 Effect of subchronic oral administration of aqueous stem bark extract of K senegalensis on liver function parameters of Albino rats

Test 0mgkg bw 400mgkg bw 800mgkg bw 1200mgkg bw 1600mgkg bw 2000mgkg bwT protein (gdL) 956 plusmn 064 909 plusmn 022 830 plusmn 035

b804 plusmn 022

b798 plusmn 018

b759 plusmn 036

b

Albumin (gdL) 531 plusmn 044 408 plusmn 021b

405 plusmn 021b

399 plusmn 033b

424 plusmn 022b

435 plusmn 030b

Globulin (gdL) 416 plusmn 023 485 plusmn 024b


309 plusmn 029b

A G ratio 132 plusmn 022 089 plusmn 010b

096 plusmn 014b

098 plusmn 010b

112 plusmn 012 149 plusmn 016

AST(UI) 5244 plusmn 250 5415 plusmn 287 5960 plusmn 260b

5807 plusmn 335b

6124 plusmn 361b

8406 plusmn 363b

ALT (UI) 3254 plusmn 250 3758 plusmn 326 4417 plusmn 310b

5153 plusmn 300b

5322 plusmn 283b

6018 plusmn 340b

ALP (UI) 12474 plusmn 872 12641 plusmn 1256 13542 plusmn 1225b14943 plusmn 680

b19840 plusmn 260

b28793 plusmn 5180

b

T BlL (mgdL) 117 plusmn 061 192 plusmn 089 213 plusmn 071b

172 plusmn 091b

396 plusmn 060b

426 plusmn 049b

C BIL (mgdL) 091 plusmn 036 037 plusmn 021b

076 plusmn 029b

042 plusmn 017b

140 plusmn 029b

147 plusmn 027b

U BIL (mgdL) 026 plusmn 025 146 plusmn 036b

136 plusmn 032b

129 plusmn 043b

245 plusmn 047b

288 plusmn 056b



33 Effect of Subchronic Oral Administration of Aqueous StemBark Extract of K senegalensis on Kidney Function Parametersof Rats The effect of aqueous stem bark extract ofK senegal-ensis on serum urea creatinine potassium ion and sodiumion is shown in Table 2 The administration of the extractincreased the serum concentration of urea and potassium ionsignificantly (119875 lt 005) in all the different dose groups whencompared to their corresponding control valuesTherewas anobserved corresponding increase in the values of creatinineand sodium levels in all the dose groups with significant(119875 lt 005) difference at the 1600mgkg and 2000mgkg bodyweight dose group

34 Effect of Subchronic Oral Administration of Aqueous StemBark Extract of K senegalensis on Liver Function Parameters ofRats Theeffect of subchronic oral administration of aqueousstem bark extract on serum total protein albumin globulinalbuminglobulin ratio (A G) some transaminases activi-ties alkaline phosphatase activity and bilirubin is presentedin Table 3 The results indicated a significant (119875 lt 005)reduction in the values of total protein and albumin in a dosedependentmannerThe observed significant (119875 lt 005) effecton globulin and albuminglobulin ratiowas however not dosedependent The effect of the extract resulted in a significant(119875 lt 005) increase in AST ALT and ALP activities whencompared to their corresponding control values Similarlythe extract exerted a significant (119875 lt 005) decrease in totalbilirubin conjugated bilirubin and unconjugated bilirubinwhen compared to their corresponding control values

35 Effect of Subchronic Oral Administration of Aqueous StemBark Extract of K senegalensis on Haematological Indices ofAlbino Rats Results on the effect of sub-chronic oral admin-istration of aqueous stem bark extract of K senegalensis onhaematological indices are presented in Table 4 The resultsdemonstrate that the extract at the different experimentaldoses had no significant (119875 gt 005) effect on the haemato-logical indices

36Histopathology The livers of animals in the control group(0mgkg) showed typical hepatolobular architecture consist-ing of a central vein with radiating cords of hepatocytes sep-arated by sinusoids portal areas composed of the portal veinhepatic artery and bile duct were situated at the peripheryThe hepatocytes were polygonal in shape with centrallightly stained nucleus and clear nucleolus few binucleatedcells were also present and the cytoplasm was regularlydistributed without vacuolations In the treated dose groups(400 800 1200 1600 and 2000mgkg bw) the general hepa-tolobular architecture of the liverwas deranged therewas lossof radial arrangement of hepatocytes and sinusoids the num-ber of binucleated cells was increased inflammatory cellsespecially lymphocytes were found infiltrating around theportal track areas of necrosis were found around the centralvein

Examination of the liver histological preparationsobtained from all the experimental treated groups showedcomparable changes as observed in Figures 2(a) to 2(f)


Table 4 Effect of subchronic administration of aqueous stem bark extract of K senegalensis on haematological indices of Albino rats

Dose mgkgbody weight PCV () WBC

(times109 L) Neutrophils () Lymphocytes() Eosinophils () Monocytes () Platelets (times109 L)

0 4167 plusmn 250 380 plusmn 076 4767 plusmn 252 4767 plusmn 306 333 plusmn 252 233 plusmn 153 10667 plusmn 577






Values are expressed as mean plusmn SD 119899 = 5The level of significance was set at119875 lt 001when compared with control at 0mgkg body weight Dunnettrsquos multiplecomparison testPCV packed cell volume WBC white blood cell

(a) (b)

(c) (d)

(e) (f)

Figure 2 Photomicrographs of liver sections HampE stain 400x (a) 0mgkg bw normal architecture of the liver central vein (CV) in thecenter of the hepatic lobule is filled with blood (B) Hepatocytes arranged in the form of cords are rounded in a polyhedral shape andradiate peripherally they show the nucleus with a clear nuclear membrane and nucleolus (NO) cords are separated by sinusoids (S) withKupffer cells (b) 400mgkg bw deranged architecture of the liver extensive ballooning (BL) of hepatocytes with hyperchromatic nuclei andnucleoli are not clearly seen multiple binucleated cells (BN) and sinusoidal arrangement are also deranged observed centrilobular necrosis(NC) (c) 800mgkg bw distorted architecture of the liver with ballooning degeneration of hepatocytes (H) Observed hyperchromaticnuclei (N) and nucleoli are not clearly seen multiple binucleated cells (BN) and sinusoidal (S) arrangement also seems to have beenderanged Periportal (PT) inflammation consisting of collection of lymphocytes (L) around the bile duct (BD) portal vein (PV) and hepaticartery (HA) (d) 1200mgkg bw feathery ballooning of hepatocytes with sinusoidal (S) arrangement being deranged portal triaditis (PT)showing inflammation consisting of a collection of lymphocytes around the bile duct (BD) (e) 1600mgkg bw deranged architecture of liverwith hepatocyte vacuolation ballooning (BL) of hepatocytes with nucleoli not clearly seen multiple binucleated cells (BN) and sinusoidalarrangement have been deranged periportal triaditis (PT) showing distorted triads and inflammation consisting of collection of lymphocytes(L) around the hepatic artery (HA) observed hepatic necrosis (NC) (f) 2000mgkg extensive ballooning (BL) of hepatocytes with multiplebinucleated dilation with centrilobular necrosis (NC)


(a) (b)

(c) (d)

(e) (f)

Figure 3 Photomicrographs of kidney sections HampE stain 400x (a) 0mgkg bw observed normal proximal and distal tubules (DT)glumerulus (G) blood vessels and interstition Podocyte (P) urinary pole (UP) and corpuscular space (CS) appear within normal range (b)400mgkg bw proximal (PT) and distal tubule (DT) glomerulus (G) blood vessels and interstition Podocyte (P) and corpuscular space (CS)appear within normal range (c) 800mgkg bw normal architecture of a kidney section cross-section of an arteriol (A) and a closely packedrenal tubule is observed (d) 1200mgkg bw normal distal tubule (DT) glomerulus (G) blood vessels and interstation (e) 1600 mgkg bw aclosely packed renal tubule is observed and corpuscular space (CS) appears within normal range (f) 2000mgkg bw normal proximal tubule(PT) glomerulus (G) and corpuscular space (CS) appear within normal range

The histological preparations of the kidney and heart ofthe rats based on the subchronic toxicity study of aqueousstem bark extract of K senegalensis indicated that they hadnormal architecture (Figures 3 and 4)The observed changeshowever in all the treated groups were restricted to only mildcongestion and were not dose dependent

4 Discussion

The LD50of aqueous stem bark extract of K senegalensis was

found to be greater than 5000mgkg it may therefore beconsidered nontoxic although this does not predict the lethal

dose in humans it however provides a guide for choosing thedose for use in sub-chronic studies

Bodyweight is normally investigated as a sensitive indica-tor of chemically induced changes to organsThe comparisonbetween the ratiometric differences of organ body weight ofthe control group and the treated groups has been used toevaluate the toxic effect of the test substance [23]The result ofthis study shows that sub-chronic administration of K sene-galensis does not affect the anatomical proportion (ratios ofkidney body weight and heart body weight) as compared tonormal control this may be that the defensive mechanismof the animal has not been overcome andor may be thatthe dose has not accumulated sufficiently to manifest any


(a) (b)

(c) (d)

(e) (f)

Figure 4 Photomicrographs of heart sections HampE stain 400x (a) 0mgkg bw normal architecture of the heart section A cardiac myocyte(CM) appears within the normal limits with nuclei (NC) present A normal intercalation disk with mild congestion (b) 400mgkg bw theheart section appears within normal limits with visible cardiac myocyte and nuclei (NC) observed normal intercalation disk with mildcongestion (c) 800mgkg bw normal architecture of the heart section A cardiac myocyte (CM) appears within the normal limits with nucleipresent (NC) (d) 1200mgkg bw heart sections cardiac myocyte nuclei (NC) and intercalation disk appear within the normal limits (e)1600mgkg bw normal architecture of the heart section (f) 2000mgkg bw normal architecture of the hearth section

significant change However the significant increase of theratio of liver body weight (hepatomegaly) following theadministration of the extract may be attributed to tissueoedema (oedematous) and ballooning resulting in necrosisthis may be due to the role of the liver in detoxification andexcretion The spleen at the dose group of 2000mgkg bodyweightwas significantly (119875 lt 005) increased (splenomegaly)this could result from any one of the following hemolyticanemia cirrhosis hepatic vein obstruction and portal veinobstruction It could also have been a result of an inducedalpha-mannosidosis by the extract that is if the extract wasable to penetrate into the lysosome It is therefore worthy tomention that the higher dose levels (1600 and 2000mgkgbody weight) should be used with caution

The possibility of renal insufficiency or dysfunctionoccurring as a result of the toxic effect of the extract maybe eminent The observed (Table 2) significant increase inserum sodium ions with a concomitant significant increase inpotassium ion levels may be due to renal dysfunction result-ing from the inability of the kidney to regulate an electrolytebalance The extract may also interact with specific hormonereceptors resulting in increased production of aldosteroneand mineralocorticoids which may be responsible for theobserved hypernatraemia Furthermore the marked eleva-tion in serum urea level in this study may suggest that theextract caused renal impairment in the rats or it may be aresult of increased protein catabolism However since ureais not a complete estimation of renal function the observed


significant increase in creatinine may further buttress theextract as toxic to the kidney at the doses used in this study

Serums ALT and AST considered in this study are impor-tant and play significant role in the diagnosis of liver cytolysis[24] The estimation of the levels of protein and bilirubin isused to examine the synthetic and excretory function of theliver respectively Measurement of the activities of enzymesin tissues and body fluids plays a significant and well-known role in disease investigation and diagnosis [25] Tissueenzyme assay can also indicate tissue cellular damage longbefore structural damage can be picked up by conventionalhistological techniques Such measurement can also give aninsight to the site of cellular tissue damage as a result of assaultby the plant extract [26]

In this study the resultant increase in the activities ofserums ALT and AST may be an indication of liver cytolysisthrough which they are released into circulation ALT islocated in the cytosol of hepatocytes and the enzyme isconsidered a more sensitive marker of hepatocellular damagethan AST AST is found in the cytoplasm and mitochondriain different tissues of the heart liver kidney skeletal musclepancreas and erythrocyte [27] The aminotransferases aresignificant in amino acidmetabolism as they help in retainingamino groups during the degradation of amino acids whichare further used for the synthesis of new amino acids andhence they are involved in the biochemical regulation of theintracellular amino acid pool Also when these enzymes aremobilized from the liver into the free circulation glutamateconcentration may be affected with a resultant decrease inglutathione one-third of which is formed from glutamate[28] Detoxification is the essential role of glutathione andcells are exposed to an attack by the active metabolite when itis depleted Also a significant (119875 lt 005) change in the level oftotal bilirubin was observed (Table 2) This may indicate thatthe functional integrity of the liver has been affected by thedamage caused to the liver by the plant extract [29] Bilirubinis assayed to measure the binding excreting and conjugatingability of a hepatocyte Hence the observed increase mayindicate that a component of the plant extract might havecompeted and displaced the binding of bilirubin on albuminor the uptake of bilirubin is inhibited by the plant extractthis may also account for the increased level of unconjugatedbilirubin All these would lead to an increase in total bilirubinin the serum Extract administration produced a significantdecrease in the total protein concentration in all the dosegroups with the exception of 400mgkg body weight Hencethe aqueous stem bark extract might inhibit the synthesisof some proteins thereby resulting in the decrease in serumtotal protein It has been reported that lowprotein level resultswhen there is extensive liver damage [30] The result of thisstudy revealed significant (119875 lt 005) increase in the activityof serum alkaline phosphatase (ALP) ALP is often employedto assess the integrity of the plasma membrane of the liver[31] The significant increase in the serum ALP followingadministration of the plant extracts may be due to disruptionof the liver plasma membrane The result of this study there-fore demonstrates that K senegalensis at the experimentaldoses is nephrotoxic and should be used with caution whenadministered over a long period of time

The possibility of the extract giving rise to haemopoieticdamage malignancy allergy or compromise to the immunestatus could be ruled out since all haematological parametersassayed did not show any significant changes when comparedwith their corresponding controls

The liver being an organ of detoxification is the first organthat encounters all absorbed materials from the gastroin-testinal tract it has been shown to respond to toxicologicalinsults in a number of ways including cellular degenerationand necrosis and bile duct hyperplasia and fibrosis [32]Thesefindings are consistent with those of Garba et al [33] andMutalik et al [34] its protective property by detoxificationmay explain the preserved architecture of the kidney andheartThemild tomoderate congestion aswell as hemorrhageobserved on the histological slides could be attributed to sur-gical handling of the organs since these conditions were alsoobserved in the normal groups

5 Conclusion

These observations suggest that long term administration ofK senegalensis may be toxic and its toxicity may be organspecific

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

The second author wishes to thank the Tertiary EducationTrust Fund (TETFund) for the grant used for this research

References

[1] RW J Keay C F F Onochie and J StanfieldARevised Versionof Trees of Nigeria Clarendon Press New York NY USA 1989

[2] L S Gill Ethnomedical Uses of Plants in Nigeria University ofBenin Press Benin Nigeria 1992

[3] M Iwu Hankbook of African Medicinal Plants Pharmacognos-tical Profile of Selected Medicinal Plants CRC Press Inc 1993

[4] M Lompo J B Nikiema I P Guissou A J Moes and JFontaine ldquoThe topical anti-inflammatory effect of chloroformextracts from Khaya senegalensis stembarksrdquo PhytotherapyResearch vol 12 pp 448ndash450 1998

[5] W M Kone K Kamanzi Atindehou C Terreaux K Hostett-mannD Traore andMDosso ldquoTraditionalmedicine inNorthCote-drsquoIvoire screening of 50 medicinal plants for antibacterialactivityrdquo Journal of Ethnopharmacology vol 93 no 1 pp 43ndash492004

[6] I O Ademola B O Fagbemi and S O Idowu ldquoEvaluation ofthe anthelmintic activity of Khaya senegalensis extract againstgastrointestinal nematodes of sheep in vitro and in vivo studiesrdquoVeterinary Parasitology vol 122 no 2 pp 151ndash164 2004

[7] X M Androulakis S J Muga F Chen Y Koita B Toureand M J Wargovich ldquoChemopreventive effects of Khaya sene-galensis bark extract on human colorectal cancerrdquo AnticancerResearch vol 26 no 3 B pp 2397ndash2405 2006

[8] A El-Tahir A M Ibrahim G M H Satti T G TheanderA Kharazmi and S A Khalid ldquoThe Potential Antileishmanial


activity of some Sudanese medicinal plantsrdquo PhytotherapyResearch vol 12 pp 576ndash579 1998

[9] G A Adesida E K Adesogan D A Okorie D A H Taylorand B T Styles ldquoThe limonoid chemistry of the genus Khaya(meliaceae)rdquo Phytochemistry vol 10 no 8 pp 1845ndash1853 1971

[10] D A Mulholland B Parel and P H Coombes ldquoThe chemistryof the Meliaceae and Ptaeroxylaceae of Southern and EasternAfrica and Madagascarrdquo Current Organic Chemistry vol 4 no10 pp 1011ndash1054 2000

[11] T Narender T Khaliq K P Shweta-Reddy and R K SharmaldquoOccurrences Biosynthesis Activity and NMR spectroscopy ofD and B D ring seco-limonoids of Meliaceae familyrdquo NaturalProduct Communications vol 2 pp 203ndash221 2007

[12] J B Harborne Phytochemical Methods Chapman amp Hall Lon-don UK 1973

[13] OECD ldquoAcute Oral Toxicity-up and down procedurerdquo Guide-lines for Testing of Chemicals vol 425 pp 1ndash26 2004

[14] B T DoumasQuoted in CheesbroughM 1992 vol 1 ofMedicalLaboratory Manual for Tropical Countries ELBS CambridgeUK 2nd edition 1975

[15] K Spencer and C P Price ldquoGuidelines on standard operatingprocedures for clinical chemistryrdquo Annals of Clinical Biochem-istry vol 14 pp 105ndash115 1977

[16] S Reitman and S Frankel ldquoA colorimetricmethod for the deter-mination of serum glutamic oxalacetic and glutamic pyruvictransaminasesrdquo American Journal of Clinical Pathology vol 28no 1 pp 56ndash63 1957

[17] G K C Deutsche ldquoOptimised standard colorimetric methods(SerumAlkaline Phosphatase)rdquo Journal of Clinical Chemistry ampClinical Biochemistry vol 10 article 182 1972

[18] J K Fawcett and J E Scott ldquoA rapid and precise method for thedetermination of ureardquo Journal of Clinical Pathology vol 13 pp156ndash159 1960

[19] B Seaton and A Ali ldquoSerum creatinine levelrdquoMedical Labora-tory Scientist vol 41 pp 327ndash336 1984

[20] M Cheesbrough Medical Laboratory Manual for TropicalCountries vol 1 EESB Cambridge UK 2nd edition 1992

[21] J V Dander and S M Lewis Practical Hematology ChurchillLivingstone Edinburgh Scotland 7th edition 1991

[22] J A Kiernan Histological and Histochemical Methods Theoryand Practice Pergamon Press Oxford UK 1981

[23] J M Peters and E M Boyd ldquoOrgan weights and water levels ofthe rat following reduced food intakerdquo Journal of Nutrition vol90 no 4 pp 354ndash360 1966

[24] M Shahjahan K E Sabitha M Jainu and C S Shyamala DevildquoEffect of Solanum trilobatum against carbon tetrachlorideinduced hepatic damage in albino ratsrdquo Indian Journal of Medi-cal Research vol 120 no 3 pp 194ndash198 2004

[25] S O Malomo ldquoToxicological implications of ceftriaxoneadministration in ratsrdquo Nigerian Society of Biochemistry andMolecular Biology vol 15 pp 33ndash38 2000

[26] J O Adebayo M T Yakubu E C Egwim V B Owoyele and BU Enaibe ldquoEffect of ethanolic extract of Khaya senegalensis onsome biochemical parameters of rat kidneyrdquo Journal of Ethno-pharmacology vol 88 no 1 pp 69ndash72 2003

[27] S W Hassan A A Umar A A Ebbo A J Akpeji and I KMatazu ldquoHepatoprotective effect of leaf extracts of Parkinsoniaaculeate L Against CCL intoxication in albino ratsrdquo Interna-tional Journal of Biochemistry vol 2 no 2 pp 42ndash48 2008

[28] L Stryer Principles of Biochemistry WH Freeman and Com-pany New York NY USA 4th edition 1995

[29] H A Edmonson and R L Peters ldquoLiverrdquo in Andersenrsquos Pathol-ogy J M Kissane Ed vol 2 pp 1096ndash1212 Mosby St LouisMo USA 8th edition 1985

[30] HWada andE E Snell ldquoEnzymatic transamination of pyridox-amine II Crystalline pyridoxamine-pyruvate transaminaserdquoThe Journal of Biological Chemistry vol 237 pp 133ndash137 1962

[31] M A Akanji ldquoEffect of chronic consumption of metabisulphiteon the integrity of the rat kidney cellular systemrdquo Toxicologyvol 81 no 3 pp 173ndash179 1993

[32] Y Saidu L S Bilbis M Lawal S A Isezuo and R A UmarldquoHematotoxicity study of the Leaf Extract of AlbiziachevalierirdquoBiochemia Medica vol 17 no 2 pp 139ndash270 2007

[33] S H Garba S Ahmadu and I A John ldquoThe effect of aqueousstembark extract of Sclerocarya birrea (Hoechst) on alcohol car-bon tetrachloride induced liver damage in ratsrdquo Pakistan Jour-nal of Biological Sciences vol 9 no 12 pp 2283ndash2287 2006

[34] SMutalik M Chetana B Sulochana P U Devi andN UdupaldquoEffect of Dianex a herbal formulation on experimentallyinduced diabetes mellitusrdquo Phytotherapy Research vol 19 no 5pp 409ndash415 2005

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


2 Materials and Methods

21 Plant Materials Stem bark of K senegalensis was col-lected from the main campus of Usmanu Danfodiyo Univer-sity Sokoto (130 211015840 1610158401015840 N and 50 51015840 3710158401015840 E) in the month ofJune 2011The plant was identified by a taxonomistMal AMUmar from the Botany Unit Department of Biological Sci-ences Usmanu Danfodiyo University Sokoto and a voucherspecimen (UDUSVS201121) was prepared and deposited inthe herbarium of the same department

22 Experimental Animals Albino male rats weighing 150ndash200 g were purchased from the Animal House UsmanuDanfodiyo University Sokoto Nigeria They were housed inmetal cages and allowed free access to a standard laboratorypellet diet (ECWA Feed Nig Ltd) and water ad libitumunder a 12-hour light-dark cycle throughout the experimentalperiod

23 Preparation and Extraction of Plant Samples Plant mate-rials were washed and air-dried at room temperature untila constant weight was obtained The samples were groundusing a pestle and mortar and the powdered samples werethen bagged and stored in plastic bags until required

The plant sample was separately placed in an Erlenmeyerflask and one liter of distilled water was added into the flaskand it was allowed to soak for aminimumof 24 hours 5wvof the plant material was prepared The mixture was filteredusing a piece of clean sterile Muslin cloth to remove debrisand then filtered on Whatman filter paper The resultantfiltrate was evaporated to dryness [12] and the percentagerecovery was calculated (133)

24 Acute Toxicity Study Acute oral toxicity was performedusing the up- and down-procedure of the Organisation forEconomic Co-operation and Development [13] This proce-dure involves the limit and main test which was also usedfor the selection of a starting dose as well as determination ofLD50of the testing material

25 Subchronic Toxicity Study Animals were divided into sixgroups of five animals each The test substance was adminis-tered orally to the rats in groups IIndashIV in a gradation of 400800 1200 1600 and 2000mgkg body weight per day for 28days while group I (control group) animals were given anequivalent volume of normal saline orally for the same periodof 28 days under the same conditions

The body weight changes were monitored weeklythroughout the experimental period On the 29th day afteran overnight fast the rats were anaesthetized in a chloroformvapour and blood samples were collected from the animals bycardiac puncture into labeled Vacutainers for biochemicaland haematological assays Subsequently the animals weresacrificed by decapitation the organs (kidney liver andheart) were removed weighed and then fixed in 10 forma-lin for histopathological examinations [13]

26 Biochemical and Haematological Analysis Serum totalprotein was determined using the biuret method cupric ionsin the biuret reagent react with the peptide bonds of the pro-tein molecule in an alkaline solution to form a blue coloredcomplex [14] Albumin was determined using the Bromocre-sol Green method albumin binds with Bromocresol Greenin an acidic medium to form a blue-green complex whosecolour is proportional to the concentration of albumin in theserum [15] Transaminase activity (aspartate aminotrans-ferase (AST) and alanine aminotransferase (ALT))was deter-mined using the Reitman and Frankel [16] method ALT wasmeasured by monitoring the concentration of pyruvatedhydrazone that is formed with 24-dinitrophenylhydrazinewhile AST was measured by monitoring the concentrationof oxaloacetate hydrazone that is formed with 24-dinitro-phenylhydrazine Alkaline phosphatase (ALP) activity wasdetermined using the colorimetric method of Rec [17] thehydrolysis of 4-nitrophenyl phosphate at alkaline pH to4-nitrophenol and inorganic phosphate is catalysed by alka-line phosphatase and the intensity of the colour is directlyproportional to the activity of ALP Urea was investigatedusing the Urease-Berthelot colorimetric method of Fawcettand Scott [18] urea in serum is hydrolysed to ammonia in thepresence of urease The ammonia formed is measured spec-trophotometrically by the Berthelot reaction Creatinine wasmeasured by the picrate method [19] creatinine reacts withpicric acid in an alkaline medium to form a yellow-red col-ored complexwhich can bemeasured spectrophotometricallyat 520 nm Serum electrolytes Na+ and K+ were measuredusing a flame emission spectrophotometer Na+ and K+ areatomised using a flame the amount of atomisation is propor-tional to the quantity of the element in the solution whileHCO3was estimated using a titrimetric method [20] Packed

cell volume (PCV) of samples was determined using micro-hematocrit centrifuge at 12000 g for 5min [21] white bloodcell (WBC) count and differentials were determined bymechanical expansion and optical magnification augmentedby supravital cell staining [21]

27 Histopathological Analysis Three animals from eachgroup were selected randomly and dissected through a cen-tral abdominal incision The kidney heart and liver sampleswere collected and immediately fixed in 10 formal saline inlabeled sample plastic bottles The tissues were dehydrated ingraded concentrations of xylene embedded in molten paraf-fin wax and sectioned at 5 120583 Tissue sections were fixed onglass slides and stained with hematoxylin and eosin for lightmicroscopy at 400x [22] Photomicrographs of some of thetissues were taken using a microscope fitted with a cameraunit

28 Statistical Analysis Data were expressed as mean plusmnstandard deviation for the given number of observationsThe results were analyzed using one-way analysis of variance(ANOVA) followed by Dunnettrsquos multiple comparison testusing GraphPad Instat Software Differences were consideredsignificant when 119875 lt 005


CVB

K

S

H

H

N

NO

BD

(a)

S

N

L

(b)



Dosemgkg bw






1600 00057 plusmn 00003 00383 plusmn 000012b


2000 00055 plusmn 00004 00398 plusmn 00017b

00031 plusmn 00005 00043 plusmn 00001b

3532 plusmn 99b



3 Results









400 487 plusmn 069b


800 492 plusmn 029b


1200 489 plusmn 037b


1600 507 plusmn 016b

4944 plusmn 942b

1408 plusmn 455b

558 plusmn 041b

2000 502 plusmn 020b

7046 plusmn 1099b

1428 plusmn 410b

621 plusmn 024b





b804 plusmn 022

b798 plusmn 018

b759 plusmn 036

b


405 plusmn 021b

399 plusmn 033b

424 plusmn 022b

435 plusmn 030b



309 plusmn 029b


096 plusmn 014b

098 plusmn 010b



5807 plusmn 335b

6124 plusmn 361b

8406 plusmn 363b


5153 plusmn 300b

5322 plusmn 283b

6018 plusmn 340b


b19840 plusmn 260

b28793 plusmn 5180

b


172 plusmn 091b

396 plusmn 060b

426 plusmn 049b


076 plusmn 029b

042 plusmn 017b

140 plusmn 029b

147 plusmn 027b


136 plusmn 032b

129 plusmn 043b

245 plusmn 047b

288 plusmn 056b



















(a) (b)

(c) (d)

(e) (f)



(a) (b)

(c) (d)

(e) (f)



4 Discussion






(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


CVB

K

S

H

H

N

NO

BD

(a)

S

N

L

(b)



Dosemgkg bw






1600 00057 plusmn 00003 00383 plusmn 000012b


2000 00055 plusmn 00004 00398 plusmn 00017b

00031 plusmn 00005 00043 plusmn 00001b

3532 plusmn 99b



3 Results









400 487 plusmn 069b


800 492 plusmn 029b


1200 489 plusmn 037b


1600 507 plusmn 016b

4944 plusmn 942b

1408 plusmn 455b

558 plusmn 041b

2000 502 plusmn 020b

7046 plusmn 1099b

1428 plusmn 410b

621 plusmn 024b





b804 plusmn 022

b798 plusmn 018

b759 plusmn 036

b


405 plusmn 021b

399 plusmn 033b

424 plusmn 022b

435 plusmn 030b



309 plusmn 029b


096 plusmn 014b

098 plusmn 010b



5807 plusmn 335b

6124 plusmn 361b

8406 plusmn 363b


5153 plusmn 300b

5322 plusmn 283b

6018 plusmn 340b


b19840 plusmn 260

b28793 plusmn 5180

b


172 plusmn 091b

396 plusmn 060b

426 plusmn 049b


076 plusmn 029b

042 plusmn 017b

140 plusmn 029b

147 plusmn 027b


136 plusmn 032b

129 plusmn 043b

245 plusmn 047b

288 plusmn 056b



















(a) (b)

(c) (d)

(e) (f)



(a) (b)

(c) (d)

(e) (f)



4 Discussion






(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





400 487 plusmn 069b


800 492 plusmn 029b


1200 489 plusmn 037b


1600 507 plusmn 016b

4944 plusmn 942b

1408 plusmn 455b

558 plusmn 041b

2000 502 plusmn 020b

7046 plusmn 1099b

1428 plusmn 410b

621 plusmn 024b





b804 plusmn 022

b798 plusmn 018

b759 plusmn 036

b


405 plusmn 021b

399 plusmn 033b

424 plusmn 022b

435 plusmn 030b



309 plusmn 029b


096 plusmn 014b

098 plusmn 010b



5807 plusmn 335b

6124 plusmn 361b

8406 plusmn 363b


5153 plusmn 300b

5322 plusmn 283b

6018 plusmn 340b


b19840 plusmn 260

b28793 plusmn 5180

b


172 plusmn 091b

396 plusmn 060b

426 plusmn 049b


076 plusmn 029b

042 plusmn 017b

140 plusmn 029b

147 plusmn 027b


136 plusmn 032b

129 plusmn 043b

245 plusmn 047b

288 plusmn 056b



















(a) (b)

(c) (d)

(e) (f)



(a) (b)

(c) (d)

(e) (f)



4 Discussion






(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












(a) (b)

(c) (d)

(e) (f)



(a) (b)

(c) (d)

(e) (f)



4 Discussion






(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)

(c) (d)

(e) (f)



4 Discussion






(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)

(c) (d)

(e) (f)










5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







5 Conclusion




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Effect of Aqueous Stem Bark Extract of ...downloads.hindawi.com/journals/jt/2013/803835.pdfKhaya senegalensis for twenty-eight days at the experimental dose resulted