Acute and sub-acute toxicity studies of the methanol extractof the leaves of Paullinia Pinnata (Linn.) in wistar

albino mice and rats

OA Adeyemo-Salami1 and JM Makinde2

Departments of Biochemistry1 and Pharmacology and Therapeutics2,College of Medicine, University of Ibadan, Ibadan, Nigeria

Afr. J. Med. med. Sci. (2013) 42,

SummaryAim: The aim of this study was to investigate thetoxicological effects of the leaves of Paullinia pinnataLinn.(PP) in rodents using Wistar albino mice and ratsas experimental models.Methodology: Acute toxicity study of the methanolextract of PP was carried out in Wistar strain albinomice using varying doses of the extract at 100, 200,400, 800, 1600, 3200, 6400, and 10,000 mg/kg bodyweight . These doses were administered orally to maleWistar albino mice with the exception of the controlgroup and observed for morbidity and mortality afterDay 1, Day 7 and Day 14. Sub-acute toxicity studywas conducted in male Wistar albino rats with varyingdoses of 50, 100, 200, 400 and 800 mg/kg body weight.These doses were administered orally once daily at 24hour intervals for 28 days and the vehicle (physiologicalsaline and Tween 80 (70:30v/v)) was administered tothe control groups in the experiments. Biochemicalanalyses were carried out on the plasma whilepathological changes in the kidneys, liver and lungs wereexamined histologically.Results: In the acute toxicity study, the mice did notshow any form of morbidity or mortality. For the sub –acute toxicity study, plasma levels of alkaline phosphatase(ALP), aspartate aminotransferase (AST), totalcholesterol and the triglycerides were significantlyelevated (p<0.05) at the 400mg/kg body weight dosage.Elevated levels of plasma ALP were also observed at800mg/kg body weight. The histopathological studyshowed that the lungs exhibited dose -dependentlymphocytic infiltrations and the pattern of occurrenceof lesions observed in the liver was at a frequency ofone rat per group at the 400 and 800mg/kg body weightdoses.Conclusion: The methanol leaf extract of Paulliniapinnata (Linn.) is well tolerated when orallyadministered at a dose of 200mg/kg body weight buttoxic at higher doses.

Keywords: Acute-toxicity, sub- acute toxicity,biochemical analyses, Paullinia pinnata Linn.,histopathology, phytochemical screening

Correspondence: Oluwatoyin A. Adeyemi-Salami, Departmentof Biochemistry, College of Medicine, University of Ibadan,Ibadan, Nigeria. E-mail: soluwatoyin @yahoo.com

SummaryAim: The aim of this study was to investigate thetoxicological effects of the leaves of Paulliniapinnata Linn.(PP) in rodents using Wistar albino miceand rats as experimental models.Methodology: Acute toxicity study of the methanolextract of PP was carried out in Wistar strain albinomice using varying doses of the extract at 100, 200,400, 800, 1600, 3200, 6400, and 10,000 mg/kg bodyweight . These doses were administered orally tomale Wistar albino mice with the exception of thecontrol group and observed for morbidity andmortality after Day 1, Day 7 and Day 14. Sub-acutetoxicity study was conducted in male Wistar albinorats with varying doses of 50, 100, 200, 400 and 800mg/kg body weight. These doses were administeredorally once daily at 24 hour intervals for 28 days andthe vehicle (physiological saline and Tween 80(70:30v/v)) was administered to the control groupsin the experiments. Biochemical analyses werecarried out on the plasma while pathological changesin the kidneys, liver and lungs were examinedhistologically.Results: In the acute toxicity study, the mice did notshow any form of morbidity or mortality. For the sub–acute toxicity study, plasma levels of alkalinephosphatase (ALP), aspartate aminotransferase(AST), total cholesterol and the triglycerides weresignificantly elevated (p<0.05) at the 400mg/kg bodyweight dosage. Elevated levels of plasma ALP werealso observed at 800mg/kg body weight. Thehistopathological study showed that the lungsexhibited dose -dependent lymphocytic infiltrationsand the pattern of occurrence of lesions observed inthe liver was at a frequency of one rat per group atthe 400 and 800mg/kg body weight doses.Conclusion: The methanol leaf extract of Paulliniapinnata (Linn.) is well tolerated when orallyadministered at a dose of 200mg/kg body weight buttoxic at higher doses.

IntroductionPaullinia pinnata (Linn.) (PP) is of the familySapindaceae. There is no known common name.Some of the local names include Yoruba : Kakansenla,

Edo: Aza, Hausa: Goorondoorinaa, Sierra Leone-Kono: Kamakagu and Togo- AnyiAnufo: Tolundi.Paullinia pinnata is a woody/ subwoody climber ofdamp sites in the savannah zone. The leaf is claimedto be a panacea for various ailments [1] and is takencopiously and widely throughout the Regions of Westand Central Africa in variable forms for treatingabdominal colic, diarrhea, dysentery and anemia aswell as gynaecological usages together with otherlocal spices [2]. It is also used as fish poison [2,1].

Various investigations have shown in recentwork the presence of alkaloids, flavonoids, tannins,saponins, cardiac glycosides and reducing sugars[3,4]. Osarenmwinda et al [3] reported the dose-dependent antidiarrhoeal property of the methanolic leafextract. Ior et al [4] evaluated the analgesic and anti-inflammatory activities of the ethanolic extract of theleaves in mice and rats and reported significant analgesicand anti-inflammatory effects. Maje et al [5] reportedthe effects of ethanolic leaf extract on early, repositoryand established malaria infections using Swiss albinomice. Extracts of the leaves and root have also beenreported to have anti- oxidant properties [6].

This study was designed to investigate theacute and sub-acute toxicity of the leaves of PP whichhave been unexplored using phytochemical,physiological, histopathological and biochemicalanalyses in Wistar albino mice and rats.

Materials and methodsAnimal experiment ethical reviewThe methods for the preparation of the animals, modeof administration and the group size was incompliance with International scientific standardprocedures: for Acute toxicity study:-Kennedy et al[7] and Toskulkao et al [8], and for Sub- acute toxicitystudy:- Karrow et al [9].

ReagentsSodium hydroxide (Sigma New York, U.S.A.),Alkaline Phosphatase (ALP), Aspar tateaminotransferase(AST), Alanine aminotransferase(ALT), total protein, total bilirubin, albumin, totalcholesterol, triglyceride, urea and creatinine levels inthe plasma were analyzed using appropriate Randoxbio-assay Kits (Co. Antrim, U.K.).

InstrumentsThe absorbance at different wavelengths, as specifiedfor each parameter, was measured using Beckman-Coulter DU 520 spectrophotometer (California,U.S.A.). The Olympus binocular light microscope(Tokyo, Japan) was used in observing the histologyslides of the organs and the Easylite analyzer

(Massachusett, U.S.A.) was used to assay thesodium and chloride ions in the plasma.

Plant samples: The leaves of PP were collectedfrom the Forestry Research Institute of Nigeria(FRIN), Ibadan, Nigeria. The plant was authenticatedat the same Institute and given the specimen vouchernumber FHI 106555.

Extraction and preparation of plant materialsThe leaves were air-dried, milled and extracted inabsolute methanol for 6 hours in a Soxhlet extractorover a steam bath and a 14% yield of the extractwas realized.

Phytochemical screeningThe method of Sofowora was adopted [10]. An extractof the shade dried and pulverized leaves of Paulliniapinnata (Linn) was prepared by macerating 30g in450ml of redistilled methylated spirit, filtered and theprocess repeated until all soluble compounds had beenextracted. This was indicated by loss of colour of filtrate.The total extract from the plant part was evaporated todryness in vacuo at about 450c and further dried toconstant weight at the same temperature in a hot-airoven. Portions of it were used to test for the followingplant constituents alkaloids, tannins, saponins,anthraquinones and cardiac glycosides.

Preparation of animals for acute and Sub- acutetoxicity studiesFifty-four (54) male Wistar albino mice whose weightsranged from 18- 25g were used for the acute- toxicitystudy and 36 male Wistar albino rats whose bodyweights ranged from 140- 190g were used for the sub-acute toxicity study. They were obtained from theCentral Animal House, College of Medicine Universityof Ibadan. They were kept in the Animal House,Department of Biochemistry, University of Ibadan,Nigeria for one month to acclimatize with free accessto feed (obtained from Guinea Feed livestock Limited,Nigeria) and laboratory water before starting theexperiment. They were weighed and matched intogroups of 6 animals each. The 12-hour light/ 12-hourdark cycle was maintained.

Acute-toxicity studyEight different doses of the methanolic extract ofthe leaves were administered orally to the mice afteran overnight fast. The doses were 100, 200,400, 800,1600, 3200, 6400 and 10,000mg/kg bw. The vehicle(physiological saline with Tween 80 (70:30 v/v)) wasadministered to the control group. The animals werethen observed as follows: after Day 1, Day 7 and

OA Adeyemo-Salami and JM Makinde

Day 14 (delayed toxicity) for gross behavioural(reflexes, restlessness, sleeping pattern, locomotivedisability) / physiological (restlessness, loss of hair,redness of eye/ blindness) changes and mortality [7,8].

Sub-Acute Toxicity StudyThe animals were fasted for 14 hours. Five differentdoses of the methanolic extract of the leaves were thenadministered orally once daily to the rats at 24 hoursinterval for 28 days. The doses were 50, 100, 200, 400and 800 mg/kg body weight. The vehicle (physiologicalsaline with Tween 80 (70:30 v/v)) was administered inthe same manner to the control group. The weights ofthe animals were monitored weekly throughout the periodof study. On day 29, after the animals were fasted initiallyfor 12 hours, they were made inactive by cervicaldislocation. The animals were then cut open and bloodwas collected from the heart using a needle and 5mlsyringe into lithium heparin and ethylene diamine tetraacetic acid (EDTA) specimen bottles. The liver, lungsand kidneys from each animal were promptly removedinto chilled 0.9% NaCl solution. The organs were thenblotted using Whatman No.2 filter paper before weighingwith a Mettler balance [9].

Preparation of plasmaPlasma was separated by spinning the blood samplesin the lithium heparin and EDTA bottles at 3000 rpmfor 10 mins using an MSE bench top centrifuge(England, U.K.). The plasma was removed andcollected into plain bottles.

Alanine Aminotransferase (ALT) DeterminationBased on the colorimetric method of Reitman andFrankel [11] plasma samples from heparinised bottleswere analyzed using glutamic pyruvic transaminaseRandox Bioassay Kit. This method measures the activityof plasma ALT by monitoring the absorbance of pyruratehydrazone formed at 540nm. The activity of ALT in theplasma was obtained from a standard reference tableof ALT activity supplied with the Randox kit.

Aspartate Aminotransferase (AST) DeterminationAlso based on the colourimetric method of Reitman andFrankel [11], plasma samples from heparinised bottleswere analyzed using glutamic oxaloacetic transaminaseRandox bioassay kit. The method measures the activityof plasma AST by monitoring the absorbance ofoxaloacetate hydrazone formed at 540nm.

Alkaline phosphatase (ALP) DeterminationBased on the colorimetric optimized standard methodaccording to the recommendations of the DeutscheGesellschaft Klinische Chemie [12], plasma samples

from heparinised bottles were analyzed using ALPbioassay Randox kits. The method uses theabsorbance of p-nitrophenol formed at 405nm tocalculate the activity of ALP in the plasma.

Total plasma protein determinationBased on the method of Henry et al [13] plasmasamples from heparinised bottles were analyzedusing Total protein Randox bioassay kits.The methodis the Biuret. The absorbance of the samples and ofthe standard against reagent blank was measured ata wavelength of 550nm.

Albumin determinationThe dye binding method utilizing bromocresol green(BCG) was used to analyze the plasma samples fromheparinised bottles [14]. The albumin-BCG-complexformed absorbs maximally at 578nm, the absorbancebeing directly proportional to the concentration ofalbumin in the plasma sample.

Bilirubin determinationThe method of Powell as reported by Varley et al [14]was used to analyze plasma samples from heparinisedbottles and absorbance was taken at 520nm.

Urea determinationUsing the method of Varley et al [14] plasma samplesfrom heparinised bottles were analyzed. Absorbancewas taken at 520nm.

Creatinine determinationPlasma samples from heparinised bottles wereanalyzed using creatinine Randox bioassay kit basedon the Jaffe’s Alkaline Picrate method [14]. Theintensity of the red tautomer of creatinine picrateformed is measured at 520nm.

Sodium Ion and chloride Ion determinationUsing the Easylite Analyzer plasma samples fromheparinised bottles were analyzed for the sodium andchloride ion content. The operation of this instrument isbased on the principle of direct- ion selective electrode.

Total cholesterol determinationBased on the methods of Richmond [15], Study GroupEuropean Atherosclerosis Society [16] and Trinder[17], plasma samples from EDTA bottles wereanalyzed using Total Cholesterol Randox bioassaykits. The absorbance of the quinoneimine formed inthe presence of peroxidase is read at 500nm.

Triglyceride determinationBased on the methods of Jacobs and Denmark[18],and Trinder [17], plasma samples from EDTAbottles were analyzed using the Triglyceride Randox

Toxicity studies paullinia pinnata leaves

bioassay Kit. The absorbance of the quinoneimineformed from the reaction of hydrogen peroxide, 4-amino phenazone and 4- chlorophenol in the presenceof peroxidase is read at 500nm.

Histological analysisSectioning technique was employed to process andmount the organ sections on microscope slides.Briefly, the tissues were fixed using 10% formalin.The fixed tissues were then subjected to dehydrationby transferring the tissue gradually through differentgrades of alcohol starting from 30% alcohol toabsolute alcohol. The tissues were then de-alcoholizedusing xylene. They were then embedded in paraffinwax following which the tissues were then blockedusing moulds. The blocks were trimmed and thensubjected to sectioning using a microtome. Thesections were dewaxed in xylene and hydratedthrough different percentage grades of alcohol. Theywere then stained in British haematoxylin. Excessstain was washed off in water and the sections werethen differentiated in 70% acid alcohol. The organsections were counter stained in alcoholic eosin anddehydrated through increasing percentage grades ofalcohol. They were then cleared in xylene andmounted in Canada balsam on slides. The cover slipswere fixed and the slides were labeled.

Statistical analysisThe data were statistically evaluated using one wayanalysis of variance (ANOVA). All the results wereexpressed as mean ± SE (standard error) and p <0.05was regarded as significant.

ResultsThe phytochemical screening presented in Table 1shows the presence of alkaloids, tannins and cardiacglycosides, and the absence of saponins andanthraquinones (both free and combined) in the leavesof PP.

Table 1: Phytochemical screening

Test Result

Alkaloids1% Hydrochloric acid +veThin layer chromatography +veSaponins: Frothing test -veTannins: Ferric chloride reagent +veAnthraquinones:(a) Free -ve(b) Combined -veCardiac glycosides:Kedde test +veKeller –Kiliani test +ve

In the acute – toxicity study presented in Table 2 themice showed no gross behavioural changes whichincluded no change in reflexes, no restlessness, nochange in sleeping pattern and there was nolocomotive disability. Also the animals exhibited nophysiological changes which included no loss of hairand no redness of eyes or blindness. Mortality wasnot observed after Day 1, Day 7 and Day 14.

Over the period of the 28 days treatment ofthe male Wistar albino rats, weight of the animalsmonitored weekly showed that the rate of percentageweight gained weekly decreased in a dose –dependentmanner in the treatment groups compared to thecontrol. The group treated with the 800 mg/kg body

Table 2: Acute toxicity study in Wistar albino mice

ParametersDose(mg/kg) Reflexes Restlessness Sleeping Locomotive Loss of Redness of Mortality

pattern disability hair eyes/ blindnessControl No change Not No change Not Not Not Not

observed observed observed observed observed

100 ,, ,, ,, ,, ,, ,, ,,200 ,, ,, ,, ,, ,, ,; ,,400 ,, ,, ,, ,, ,, ,, ,,800 ,, ,, ,, ,, ,, ,, ,,1,600 ,, ,, ,, ,, ,, ,, ,,3,200 ,, ,, ,, ,, ,, ,, ,,6,400 ,, ,, ,, ,, ,, ,, ,,10,000 ,, ,, ,, ,, ,, ,, ,,

Note: n= 6

OA Adeyemo-Salami and JM Makinde

weight dose showed the least rate of percentageweight gained over the period of study (23.0%, Fig.1). Apart from the statistically insignificant decreasedweight gained, no change in morphology or grossbehavioral changes was observed and there was noincidence of mortality by the termination of the study.The percentage weight of the vital organs; kidneys,lungs and liver, did not vary significantly from that ofthe control rats although there was a dose dependentincrease (Table 3).

Further investigations into the alterations in theplasma concentrations of certain biochemicalconstituents and various bio- markers which areknown to be present in various organs were carriedout. In the liver, this includes total proteins, totalbilirubin, albumin, cholesterol and triglyceride levels

in the plasma (Tables 4&5). The sub-acute toxicitystudy showed that the plasma levels of total proteins,total bilirubin, and albumin were not significantlyelevated in the treatment groups (Table 4). Plasmacholesterol and triglyceride levels in the group treatedwith a dose of 400mg/kg body weight (80.00± 4.97mg/dl and 99.75± 8.61 mg/dl respectively) increasedsignificantly (p <0.05) compared to that of the controland the other treatment groups (Table 5).

Furthermore, the plasma levels of the enzymes; ALP,ALT and AST (Table 6) which are also indicators forliver function, showed that the plasma ALT activity wasnot significantly affected by the various doses oftreatment and the increased activity at 400 mg/kg bodyweight dose (128.25± 30.16 IU/L) was not significant.

Toxicity studies paullinia pinnata leaves

Table 3: Percentage weight of the organs relative to thebody weight in the treated and control Wistar albino rats

Dose Lungs (% Liver (% Kidneys(%(mg/kg) body weight) bodyweight) body weight)

Control 0.58 ± 0.11 3.67 ± 0.30 0.64 ± 0.0350 0.60 ± 0.02 3.50 ± 0.08 0.61 ± 0.04100 0.62 ± 0.03 3.52 ± 0.28 0.66 ± 0.02200 0.69 ± 0.05 3.99 ± 0.34 0.70 ± 0.06400 0.76 ± 0.04 4.11 ± 0.05 0.78 ± 0.03800 0.83 ± 0.07 4.07 ± 0.07 0.75 ± 0.03

Note: n = 6

Table 4: The plasma concentrations of bilirubin, totalproteins and albumin in treated and control Wistar albinorats

Dose Total Bilirubin Total Protein Albumin(mg/kg) (mg/dl) (mg/dl) (mg/dl)

Control 0.08 ± 0.05 6.78 ± 0.25 2.45 ± 0.0350 0.05 ± 0.03 6.13 ± 0.29 2.43 ± 0.10100 0.10 ± 0.04 6.68 ± 0.17 2.60 ± 0.04200 0.20 ± 0.04 5.73 ± 0.17 2.30 ± 0.13400 0.13 ± 0.03 6.73 ± 0.36 2.58 ± 0.11800 0.10 ± 0.06 6.13 ± 0.42 2.15 ± 0.18

Note: n = 6

Table 5: The plasma lipid profile in the treated and controlWistar albino rats

Dose(mg/kg) Cholesterol Triglyceride(mg/dl) (mg/dl) (mg/dl)

Control 60.25 ± 1.65 67.75 ± 8.8750 55.00 ± 6.78 69.50 ± 15.47100 71.75 ± 4.37 72.75 ± 9.13200 68.00 ± 6.67 76.75 ± 11.63400 80.00 ± 4.97* 99.75 ± 8.61*800 66.00 ± 4.00 69.00 ± 10.85

Note: n=6* -significant at p <0.05

Plasma AST levels in the group treated with the dose of400 mg/kg body weight (430.00± 15.82 IU/L) variedsignificantly (p< 0.05) from that of the control and ofthe other treatment groups (Table 6). Also, the ALPlevel in the groups treated with 400 and 800 mg/kg bodyweight doses (646.75± 32.95 and 649.15± 71.27 IU/Lrespectively) varied significantly (p <0.05) from that ofthe control (499.50± 26.39 IU/L) and the other treatmentgroups (Table 6).

The plasma creatinine levels of the treatmentgroups did not vary significantly from that of thecontrol (Table 7). This was also observed in the

Table 6: The activities of some enzymes in treated and control Wistar albino rats

Dose (mg/kg) Alkaline Phosphatase Aspartate amino Alanine amino(ALP)IU/L transferase transferase

(AST) IU/L (ALT)IU/L

Control 499.50 ± 26.39 202.50 ± 7.54 85.75 ± 6.7450 512.00 ± 49.09 300.25 ± 88.35 93.75 ± 11.63100 587.50 ± 16.26 252.00 ± 11.97 90.75 ± 3.09200 440.75 ± 15.82 324.75 ± 71.02 91.50 ± 6.12400 646.75 ± 32.95* 430.00 ± 15.82* 128.25 ± 30.16800 649.15 ± 71.27* 222.50 ± 13.03 78.50 ± 8.17

Note: n=6* -significant at p <0.05

Table 7: The Renal function indices in the treated and control Wistar albino rats

Dose (mg/kg) Creatinine(mg/dl) Urea(mg/dl) Sodium ion(mg/dl) Chloride ion(mg/dl)

Control 0.53 ± 0.03 44.17 ± 3.99 128.08 ± 1.54 97.80 ± 1.0350 0.50 ± 0.00 40.17 ± 3.36 127.20 ± 6.48 97.75 ± 4.15100 0.48 ± 0.03 46.82 ± 3.78 128.50 ± 3.17 100.28 ± 1.77200 0.43 ± 0.03 44.51 ± 3.80 118.51 ± 5.01 92.55 ± 5.19400 0.53 ± 0.06 45.28 ± 4.98 127.53 ± 2.58 100.28 ± 1.03800 0.45 ± 0.06 61.66 ± 13.40 126.93 ± 1.59 101.68 ± 0.45

Note: n = 6

OA Adeyemo-Salami and JM Makinde

concentrations of urea, sodium ion and chloride ionin the plasma. The results show that these were notaffected by the various dosages of treatment andthe observed increase in urea concentration at the800 mg/kg body weight dose (61.66± 13.40) was notstatistically significant.

DiscussionThe leaves of PP are being used and taken copiouslyin various forms in the treatment of various ailments

and diseases. Investigations need to be carried outto establish its toxic level especially when it is knownto also be used for lethal purposes [1,2].

The phytochemical screening of the driedpulverized leaves of PP was carried out using the

method of Sofowora [10] and this revealed thepresence of alkaloids using 1% hydrochloric acid andthin layer chromatography. The re-distilled methylatedspirit extract of the leaves also tested positive to ferric

Fig. 4: Section of the lung (right) showing pulmonary emphysema observed only in the group treated with the 800 mg/kg bodyweight dose. (Mag. X 100).

Fig. 2: Control liver (left).Section of the liver (right) showing periportal lymphocytic infiltration in one of the rats each treatedwith 400 and 800mg/kg body weight doses (Mag. X250).

Fig. 3: Representative section of the lung for the control group (left). Section of the lung (right) showing marked diffuse hyperemiaand thick blood vessel associated lymphocytic aggregates in the lung observed in the groups treated with the 400 and 800 mg/kgbody weight doses (Mag. X 250).

Toxicity studies paullinia pinnata leaves

chloride reagent thus indicating the presence oftannins. Using the Kedde and Keller- Kiliani tests, thepresence of cardiac glycosides was observed. However,the presence of saponins and anthraquinones were notobserved in the leaves (Table 1). This is similar to theresults of Ior et al [4].

The acute – toxicity study showed that thetreatment did not cause any physiological (loss ofhair, redness of eyes or blindness) or gross behavioral(reflexes, restlessness, sleeping pattern, locomotivedisability) changes, and it did not result in mortalityeven at a dose of 10,000 mg/kg body weight (Table2). This implies that the leaves of PP may be tolerableto the metabolic system of the mice even when takenin large amounts.

For the sub –acute toxicity study, thepercentage weight of the vital organs; kidneys, lungsand liver, did not vary significantly from that of thecontrol rats although a dose dependent increase wasobserved in all the organs except the kidneys (Table3). This suggests that the methanolic extract of theleaves may not have any marked deleterious effecton these organs and therefore on the metabolicsystem of the animals even at a dose of 800 mg/kgbody weight.

Plasma cholesterol and triglyceride levels inthe group treated with a dose of 400 mg/kg bodyweight increased significantly (p <0.05) comparedto that of the control (Table 5). This shows impairmentin the function of the liver. Studies have shown thatthe impairment of liver catabolic processes forchylomicron and Very low density lipoprotein(VLDL) remnants result in an increasedconcentration of both cholesterol and triglycerides inthe plasma [21]. This could be the reason for theincrease in cholesterol and triglyceride concentrationsin the plasma of the group treated with a dose of 400mg/kg body weight (Table 5). This may also be as aresult of damage or inhibition of the activity of thechylomicron remnant- and VLDL remnant- receptorsites, either directly or indirectly in the liver and inparticular, the hepatocytes. Statistically significantelevations of cholesterol and triglyceride in the plasmawere not observed at the 800 mg/kg body weightdose. This may be as a result of unavailability of thecompounds in the extract at this dose to carry outmore damage in the liver since they are likely to beinvolved in masking or inhibiting the enzymes involvedin the absorption and digestion of lipids.

Increased plasma activities of ALT and ASTare indicators of hepatocellular necrosis, especiallyALT [20,22]. Since the activity for ALT did not varysignificantly in the treatment groups, it implies thatthe liver is not affected by the treatment doses.

However, activities of ALP in the plasma weresignificantly increased at the 400 and 800 mg/kg bodyweight doses (Table 6). ALP is an indicator ofcholestasis in the liver [22]. Increased activity of ASTis also known to be due to damage done to otherorgans apart from the liver [20]. Therefore increasedactivity of this enzyme at the 400mg/kg body weightdose implies that some other organ(s) and probablythe liver were affected at this dose.

The observed differences in the activities ofthese enzymes could be that at the 800 mg/kg bodyweight dosage, which is twice the 400 mg/kg bodyweight dose, the concentration of the AST and ALTreleased into circulation have their active sites beingpartially masked or inhibited by the compounds whichare readily available at this dose and are present inthe leaf extract. This may result in the observedinsignificant decreased activity of ALT and AST inthe plasma at this dose. This would need to be furtherinvestigated.

The bio-markers for renal function arecreatinine, urea, sodium ions and chloride ions [23,24, 25]. The results show that these were not affectedby the various dosages of treatment. This furthershows that the methanolic extract of the leaves ofPP did not have any obvious effect on renal function.Results of serum/ plasma enzyme tests are to becomplimented by histological studies [20].

The histological findings complimented theresults of the test of the plasma enzymes and theinterpretation. There was a dose- dependentoccurrence of the degree of lesions in the treatmentgroups with the group treated with 800 mg/kg bodyweight showing the highest occurrence of lesions asa result of toxicity in the lungs but not the liver (Figs.2-4). This further supports the finding that AST ispresent in a wide variety of tissues apart from theliver [20].

Therefore the lesions, which are an indicationof toxicity in the lungs may be responsible for thesignificant increase in the activity of AST. Increasedactivity of ALP at the 400mg/kg body weight and800mg/kg body weight doses are reflected in theobserved lesions in the liver at these dosages . Nolesions and therefore no toxic effects were observedin the sections of the kidneys among the treatmentgroups and the control.

ConclusionA tolerable dose, for the oral administration of themethanolic extract of the leaves of Paullinia pinnata(Linn.), would be 200 mg/kg body weight which canbe converted to 14g/70kg (the average weight of an

OA Adeyemo-Salami and JM Makinde

adult). The findings in this work are a contributionto scientific knowledge of Paullinia pinnata (Linn.)

AcknowledgementsI wish to thank Professors O.G. Ademowo andCatherine A. Falade both of the Institute ofAdvanced Medical Research & Training (IAMRAT),College of Medicine, University of Ibadan, Nigeriawhon proof- read this manuscript. Late Prof. M.A.Fafunso of the Department of Biochemistry, Collegeof Medicine, University of Ibadan, Nigeria. Mr.Oladoyinbo, a Medical Laboratory Scientist of theDept. of Chemical Pathology, College of Medicine,Ibadan, Nigeria who provided the equipment for thebiochemical analyses used in this study.

Drs. Emipke and Alaka of the Faculty ofVeterinary Medicine, and Prof AAU Akang of theDept. of Pathology, College of Medicine, Universityof Ibadan, Nigeria for the interpretation of thehistological slides. Using the Third WorldOrganization for Women in Science (TWOWS)Postgraduate Training Sandwich Fellowship awardedto me in the year 2003 I conducted a part of theexperiments at the Hussein Ebrahim Jamal (H.E.J.)Research Institute of Chemistry, Karachi

References1. Burkill H.M. The useful plants of West Tropical

Africa, Families S- Z, Vol. V. Kew: RoyalGardens, 1990; 26-30.

2. Dalziel J.M. The useful plants of West TropicalAfrica. London: Crown agents for thecolonies,1937; 547-612.

3. Osarenmwinda I.P., Omonkhelin J.O. and EjiroD.: Antidiarrhoeal activity of the methanolicextract of the leaves of Paullinia pinnata Linn(Sapindaceae). The Internet Journal of health2009; 9(1) DOI: 10. 5580/10d7

4. Ior L.D. ,Uguru M.O., Olotu P.N., et al. Evaluationof analgesic and anti-inflammatory activities andphytochemical screening of the leaves extractof Paullinia pinnata (Sapindaceae). J ChemPharm Res 2011 3(4): 351-356.

5. Maje I.M., Anuka J.A., Hussaini I.M.,et al.Evaluation of the anti-malarial activity of theethanolic leaves extract of Paullinia pinnataLinn (Sapindaceae).Nig. Journ. Pharm. Sci.2007; 6 (2):67- 72.

6. Zamble A., Carpentier M., Kandoussi A., et al.Paullinia pinnata extracts rich in polyphenolspromote vascular relaxation via endothelium-dependent mechanisms. J Cardiovasc Pharmacol2006 47 (4): 599- 608.

7. Kennedy G.L., O’ Neill A.J. and Valentine R.Inhalation toxicity of dioxole and dioxolanecompounds in the rat. Drug Chem Toxicol 2001;24 (1): 1-17.

8. Toskulkao C., Chakurat L., Temcharoen P. andGlinsukon T. Acute toxicity of sterioside, a naturalsweetner and its metabolite, steviol, in severalanimal species. Drug Chem Toxicol 1997; 20 (1&2): 31-44.

9. Karrow N.A., McCay J.A., Brown R.D., et al.Evaluation of the immunomodulatory effects ofthe macrolide antibiotia, clathromycin, in femaleB6C3F1 mice: A 28- day oral gavage study. DrugChem Toxicol 2001; 24 (1): 19- 37.

10. Sofowora A. Medicinal plants and traditionalmedicine in Africa. 2nd ed. Ibadan: SpectrumBooks Limited, 1993; 150-153.

11. Reitman S. and Frankel S. A colorimetric methodfor the determination of serum glutamicoxaloacetic and glutamic pyruvic transaminases.Am J Clin Pathol 1957; 28: 56-63.

12. Recommendations GSCC (Deutsche GesellschaftKlinische Chemie).Optimized standardcolorimetric methods. J Clin Chem Clin Biochem1972; 10: 182.

13. Henry R.J., Cannon D.C. and Winkelman J.W.Clinical chemistry: Principles and techniques 2nded. London: Harper and Row, 1974; 14-84.

14. Varley H., Gowenlock A.H. and Bell M. PracticalClinical Biochemistry Vol. 1. London: WilliamHeinemann Medical Books Limited as reportedin the Manual for assays of the ChemicalPathology Unit, University College Hospital,Ibadan 1980.

15. Richmond N. Preparation and properties of acholesterol oxidase from Norcardia sp. and itsapplication to the enzymatic assay of totalcholesterol in serum. Clin Chem 1973; 19: 1350-1356.

16. Study Group, European Atherosclerosis Society.Strategies for the prevention of coronary heartdisease. A policy statement of the EuropeanAtherosclerosis Society. Eur Heart J 1987; 8: 77.

17. Trinder P. Determination of glucose in blood usingglucose oxidase with an alternative oxygenacceptor. Ann Clin Biochem 1969; 6: 24-25.

18.Jacobs N.J. and Van Denmark P.J. Thepurification and properties of the á-glycerophosphate- oxidizing enzyme ofStreptococcus faecalis 10Cl1. Arch BiochemBiophys 1960; 88: 250- 255.

19. Friedewald W.T., Levy R.I., Fredrickson O.S.Estimation of the concentration of Low –densitylipoprotein cholesterol in plasma without use of

Toxicity studies paullinia pinnata leaves

the preparative ultracentrifuge. Clin Chem 1972;18: 499.

20. Plaa G.L. and Charbonneau M. Detection andevaluation of chemically incured liver injury. InHayes AW, editor. Principles and methods oftoxicology. 3rd ed. New York: A. Raven PressLimited, 1994; 839-879.

21. Cooper A.D. Hepatic lipoprotein and cholesterolmetabolism. In Zakim D. and Boyer T.D., editors.Hepatology -A textbook of liver disease. Vol. I.Philadelphia: W.B. Saunders Com., 1990; 96-122.

22. Stolz A. and Kaplowitz N. Biochemical tests forliver disease. In Zakim D. and Boyer T.D., editors.Hepatology -A textbook of liver disease. Vol. I.Philadelphia: W.B. Saunders Com., 1990; 637.

23.Laragh T.T. and Pickering F.J. Essentialhypertension. In: Brenner BM and Rector FC,editors. Pathology of renal diseases. 4th ed. Vol.II. Philadelphia: W.B. Saunders Company,1991;1915 -1918.

24. Nduka N. Clinical Biochemistry for students ofpathology. Lagos : Longman Nigeria Plc., 1999;57-86.

25. Smith F. and Dunn A. Hypertension due to renalparenchymal disease. In: Brenner BM andRector FC, editors. Pathology of renal disease4th ed. Vol. II. Philadelphia: W.B. SaundersCompany, 1991; 1974-1975.

Received: 31/10/11Accepted: 26/03/13

OA Adeyemo-Salami and JM Makinde