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Acute toxicity studies of Croton membranaceus root extract
George A. Asarea,∗, A. Sittieb, K. Bugyeic, Ben A. Gyand, Samuel Adjeid, Phyllis Addod,Edwin K. Wiredua,e, Alex K. Nyarkod, Lydia S. Otu-Nyarkoa, David Nana Adjeia
a University of Ghana School of Allied Health Sciences (SAHS), Korle Bu, Ghanab Centre for Scientific Research in Plant Medicine Medicinal (CSRPM), Mampong, Akwapim, Ghanac Department of Pharmacology, University of Ghana Medical School, Ghanad Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Ghanae Departmenyt of Pathology, University of Ghana Medical School, Korle Bu, Ghana
a r t i c l e i n f o
Article history:Received 21 September 2010Received in revised form 25 January 2011Accepted 9 February 2011Available online 16 February 2011
Aim of the study: Croton membranaceus root and leaf extracts are used in the Bahamas to aromatizetobacco, in Nigeria to improve digestion, and in Ghana, for benign prostate hyperplasia. Despite claimsof success there is paucity of information on its toxicity. The aim of this study was to determine if Crotonmembranaceus has acute toxicity properties.Materials and methods: Roots were air-dried in a solar dryer for one week before milling. The powder wasextracted with 96% ethanol, freeze-dried and re-extracted with distilled water and freeze-dried. 15 maleSprague–Dawley rats (180–200 g) were divided equally into 2 treatment groups [low dose (LD) and highdose (HD)], plus a control group (C). LD and HD received 1500 and 3000 mg/kg b.wt. Croton membranaceusaqueous extract, respectively, one time and observed for 14 days. Haematological [Full Blood Count andhaemoglobin (Hb)], biochemical [bilirubin, alanine aminotransferase (ALA), aspartate aminotransferase(AST), total protein, albumin, globulin, alkaline phosphatise (ALP), �-glutamyltranspetidase (GGT), urea,creatinine, creatinine kinase – Muscle and Brain (CK-MB), creatinine kinase – Total (CK-R)] examinationswere performed.Results: Control group’s CK-MB (5444 ± 534 U/L) and LD group CK-MB (4014 ± 1016 U/L) were signifi-cantly different (p < 0.05). Control and the HD group CK-MB (3955 ± 1135 U/L) were significantly different(p < 0.05). Both LD and HD CK-R levels (697 ± 197 U/L and 732 ± 203 U/L, respectively), were lower thanthe control (1139 ± 220 U/L) at 48 h and 14 days (p < 0.05, p < 0.05, respectively). �-GT levels of the HD
group was 4.8 ± 0.4 U/L compared to the Control group value of 0.9 ± 0.2 U/L (p < 0.05).Conclusions: Taking all factorseral acute toxicity. However, it
Abbreviations: ALT, alanine amino transferase; AST, aspartate amino trans-ferase; AIN-93G, American Institute of Nutrition; ALB, albumin; ALP, alkalinephosphatise; ANOVA, analysis of variance; C, control; cDNA, complementarydeoxyribonucleic acid; CK-MB, creatine kinase-muscle/brain; CK-R, creatinekinase-total; CSRPM, Center for Scientific Research in Plant Medicine; EDTA-2K,ethylenediamine-N,N,N′ ,N′-tetraacetic acid, dipotassium; GAFCO, Ghana Agricul-ture Food Company; �-GT, �-glutamyltranspetidase; HCT, haematocrit; HD, highdose; HGB, haemoglobin; LD, low dose; LD50, lethal dose; LDH, lactate dehydro-genase; LYM %, lymphocytes percentage; LYM, lymphocyte count; MCH, meancorpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration;MCV, mean corpuscular volume; MPV, mean platelet volume; PDW, platelet distri-bution width; P-LCR, platelet larger cell ratio; PLT, platelet; RBC, red blood cells;RDW-CV, coefficient of variation in red cell distribution width; RDW-SD, standarddeviation in red cell distribution width; S–D, Sprague–Dawley; TP, total protein;WBC, white blood cells.
∗ Corresponding author at: Chemical Pathology Unit, Department of MedicalLaboratory Sciences, School of Allied Health Sciences, College of Health Sciences,University of Ghana, PO Box KB 143, Korle Bu, Accra, Ghana. Tel.: +233 244 627 456.
Croton comprises about 1200 species and occurs throughout thewarmer regions of the world. It is best represented in America withabout 65 species and also found in continental Africa with about125 species in Madagascar. It was introduced into Nigeria in the19th century and was described in 1864 in Ghana and Côte d’ Ivoire[Flora 47:534 (1864)]. Croton membranaceus Müll.Arg. belongs tothe Euphorbiaceae family. It is a monoecious herb or under shrubof up to 1–2 m tall. Croton membranaceus occurs in moist bush veg-etation and savanna, at low altitudes and has a limited area ofdistribution. It is apparently uncommon (Abbiw et al., 2002) buthas been cultivated in Ghana’s Aburi Botanic Gardens.
In the Bahamas, the leaves are used to aromatize tobacco and inNigeria they are used as a tonic and aromatic bitter, which improvesdigestion. The essential oil of the bark is used in aromatherapy totreat cough, fever, flatulence, diarrhea and nausea. These claimsare anecdotal and very little has been documented. The antimi-
robial activity exhibited by the Croton membranaceus root extractupports its usefulness in treating secondary bacterial infection ineasles as recently reported (Bayor et al., 2009). Furthermore, the
oot extract has been reported to exhibit markedly high cytotoxicctivities particularly against human cancer cell lines (Ayim et al.,007; Bayor et al., 2007). Finally, the root extract is used in formula-ions for the treatment and management of prostate and its relatedancer in Ghana (Mshana et al., 2000).
Six compounds from the active ethyl acetate fraction ofhis extract, include a new furano-clerodane diterpenoid, cro-omembranafuran, in addition to the known glutarimide alkaloid,julocrotine, sitosterol, sitosterol-3-d-glucoside) labdane diter-enoid, gomojoside H and dl-threitol. The root bark containscopoletin and julocrotine, as glutarimide alkaloid. It also containsalcium oxalate crystals.
Some preliminary tests on the activity of Croton membranaceusoot extract have been undertaken, but more chemical and pharma-ological research has to be done to evaluate its potential (Atakora,004). The isolation spectral data of julocrotin a glutarimide alka-
oid from Croton membranaceus has also been reported (Aboagyet al., 2002).
In this study, the acute toxicity of Croton membranaceus rootxtract was investigated.
. Materials and methods
At the commencement of the study, the protocol was reviewednd approved by the Institutional Animal Care and Use committeef the Noguchi Memorial Institute for Medical Research (NMIMR),niversity of Ghana, according to the Guidelines for Animal Studies.
.1. Plant material
Croton membranaceus roots were collected from the Gyekiti For-st Reserve area in the Eastern region of Ghana. The plant wasdentified in its vernacular names by the farmers and authenticatedy taxonomists from the Center for Scientific Research into Plantedicine (CSRPM) herbaria, where voucher specimens of the plants
ave been kept for reference purposes.
.2. Collection and extract preparation
After collection, the roots were air-dried in a solar dryer for oneeek before milling. About 1 kg of milled Croton membranaceusaterial was extracted with 96% ethanol for 24 h on a shaker at
oom temperature. Each extract was filtered and re-extracted withhe same solvent for another 24 h. The pooled extracts were concen-rated in vacuo at 50–55 ◦C before being transferred onto a freezeryer to remove traces of the solvent and water. The marc from thethanolic extractions was air-dried over-night, re-extracted withistilled water and the aqueous extract freeze-dried. The sampleas stored in a cool dry area until use.
.3. Subjects and experimental design
Fifteen (15) male Sprague–Dawley (S–D) rats (weighing about50 g) were purchased from NMIMR and housed at the CSRPM,ampong in the Eastern region of Ghana. During the acclimati-
ation period, clinical observations of the animals were conducted
s well as body weight measurement and the animals were foundealthy. The animals used for the study were assigned into groups
ncluding a control group by the stratified random method accord-ng to their body weight. S–D rats fed ad libitum a standard chowiet (AIN-93G Formulation obtained from GAFCO – Ghana).
macology 134 (2011) 938–943 939
2.4. Housing conditions
Rats were housed in plastic cages with stainless steel tops inthe animal care facility of the Center, where room temperature,humidity and ventilation were controlled. Rats were maintained ata 12-h light-cycle and were studied for 14 days. Prior to sacrifice,rats were euthanized by exsanguinations under ether anesthesia.Blood was sampled by cardiac puncture and all the visible organsand tissues were macroscopically examined and harvested.
2.5. Route of administration
The administration route was oral (gavage) in accordance withthe main route of intake of Croton membranaceus by humans formedicinal purposes.
2.6. Acute toxicity test
Five (5) S–D rats constituted a group. Thus, three groups includ-ing the control group (C) were established. A single oral LowDose (LD) of 1500 mg/kg b.wt and a single oral High Dose (HD)of 3000 mg/kg b.wt Croton membranaceus were reconstituted asan aqueous homogenous suspension containing 0.4% Tween 80.The highest dose 3000 mg/kg b.wt was selected based on previousstudies (unpublished data). The administration volume was set at1333 �l/kg b.wt. Group 1 the control group (C group) fed the nor-mal chow diet and gavaged 400 �l 0.4% Tween 80 (once). Group2, Low Dose group (LD group) and group 3, High Dose group (HDgroup) were gavaged with the extract as a once-off administrationwith the doses indicated above.
2.7. Clinical observations
The observation period was 14 days post administration. Clinicalsigns of toxidromes (such as rising fur, sluggish movement, draping,tremors, excitability, miosis, mydriasis, twitching, salivation, foodintake, morbidity, etc.) and mortality were observed while dosingand after 0.5, 1, 3, and 6 h of administration. Thereafter, twice dailyobservations were made, up until the 14th day. Body weights weremeasured before dosing on the day of administration and everymorning thereafter.
2.8. Laboratory examinations
All animals were housed individually in metabolic cages in orderto obtain freshly voided urine. Urinalysis was performed after 48 hand on the 14th day. Urine was collected and examined for pH,protein, glucose, ketone bodies, bilirubin, occult blood and uro-bilinogen.
Haematological examinations were done 48 h post extractadministration, and, on the 15th day at necropsy. Blood samplesfrom the tail of the rats (48 h) and by cardiac puncture (15th day)were collected into EDTA-2K tubes for immediate analysis using theSYSMEX hematology autoanalyzer (Kobe, Japan). Reagents for thehematology autoanalyzer were obtained from STROMATOLYZER,(WH-USA). Leukocyte count, erythrocyte count, haemoglobin con-centration, hematocrit, mean corpuscular volume (MCV), meancorpuscular haemoglobin (MCH), mean corpuscular haemoglobinconcentration (MCHC), reticulocyte ratio, platelet count and differ-ential leukocyte counts were determined.
Biochemical examinations were performed using blood col-lected into plain tubes. Blood samples were centrifuged for 5 min at3000 rpm. The following biochemical assays were performed usingSELECTRA JUNIOR VERSION 04 autoanalyzer for biochemical assays(VITAL SCIENTIFIC BV, NETHERLANDS). Total bilirubin, conjugated
940 G.A. Asare et al. / Journal of Ethnopharmacology 134 (2011) 938–943
Fig. 1. Creatinine kinase muscle/brain (CK-MB) levels post-extract administration.This figure shows the effect of Croton membranaceus aqueous extract on CK-MB at0t[p
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Fig. 2. Total creatinine kinase (CK-R) levels post-extract administration.The effectof Croton membranaceus on CK-R is seen in this figure. Significant reduction wasobserved with 1500 mg/kg b.wt administration to S–D rats compared to the Controlat 48 h and 14 days (p < 0.05) (*). Similarly at 3000 mg/kg b.wt extract administrationthe reduction in CK-R was significantly lower compared to the Control (p < 0.05) (#).This CK-R suppression persisted until day 14.
h, 48 h and 14 days. At 48 h there was a reduction in CK-MB of the LD and HDreated rats. The reduction was significant in both cases compared to the controlC vs. LD group (1500 mg/kg b.wt) p < 0.05 (*); C vs. HD group (3000 mg/kg b.wt)< 0.05 (#)].
ilirubin, aspartate amino transferase (AST), alanine amino trans-erase (ALT), total protein (TP), albumin (ALB), globulin, alkalinehosphatise (ALP), �-glutamyltranspetidase (�-GT), urea, creati-ine, lactate dehydrogenase (LDH), creatine kinase-total (CK-R) andreatine kinase-muscle/brain (CK-MB) were assayed.
The liver, kidneys, heart, lung and spleen were examined histo-ogically.
.9. Statistical analysis
The statistical analysis of the data was done using SPSS (Sta-istical Package for Social Sciences) version 17.0. Means ± SEMere determined for quatitative variables. For quantitative data
tudent’s-test was used to test for significant differences betweenwo variables. Analysis of variance (ANOVA) was used to determinehe existence of statistical significance between variables possiblyith more than two outcomes. p values ≤0.05 were considered
ignificant.
. Results
No deaths were recorded after oral administration of the extract.o physical signs of toxicity as evidenced by abnormal breathing,ovement, etc. were observed. Observation of treated animals over
he next 14 days showed no adverse effects of treatment. The oralD50 for the ethalonic extract is thus greater than 3000 mg/kg.
Urine chemistry for pH, protein, glucose, ketone bodies, biliru-in, occult blood and urobilinogen were negative for time 0 h, 48 h,nd 14 days.
CK-MB did not show significant differences among the vari-us groups at time 0 h. However, after 48 h differences betweenhe control group’s CK-MB (5444 ± 534 U/L) and the LD groupK-MB (4014 ± 1016 U/L) were statistically significant (p < 0.05).imilarly, differences between the control and the HD group CK-B (3955 ± 1135 U/L) were significant (p < 0.05). However, after 14
ays, there were no differences between the Control group and thether groups (Fig. 1).
Fig. 2 demonstrates a similar pattern as Fig. 1 for CK-R. Both LDnd HD CK-R levels (697 ± 197 U/L and 732 ± 203 U/L, respectively),ere lower than that of the control group (1139 ± 220 U/L) at 48 h.
Fig. 3. Conjugated bilirubin levels post-extract administration. Conjugated bilirubinwas significantly raised by 3000 mg/kg b.wt Croton membranaceus administrationafter 48 h. The difference between the HD group and the Control group’s conjugatedbilirbin was significant (p < 0.05) (#). However, differences were not seen at day 14.
Statistical differences were noted at 48 h between the Control andthe LD group (p < 0.05) and the Control and HD group (p < 0.05).However, the pattern persisted till the 14th day with both LD andHD CK-R values lower than the control. Furthermore, there wereno differences between the LD and HD groups.
In Fig. 3, the level of conjugated bilirubin (CB) did not varystatistically at time 0 h. Furthermore, at 48 h differences betweenthe Control CB (1.0 ± 0.2 mg/dl) and LD CB (1.1 ± 0.3 mg/dl) werenot statistically significant. However, differences between the Con-trol group and HD CB (1.4 ± 0.4 mg/dl) were statistically significant(p < 0.05). This did not persist and after 14 days no differences wereobserved between the Control group and the various groups. No sta-
tistical differences were observed between the CB levels of the LDand HD groups. Total bilirubin and unconjugated bilirubin did notshow significant statistical differences between the Control, the LDand HD groups at 48 h and 14 days. Other liver function tests such
G.A. Asare et al. / Journal of Ethnopharmacology 134 (2011) 938–943 941
Table 1Table of haematological indices of the Control group, Low Dose group (LD = 1500 mg/kg b.wt) and High Dose group (HD = 3000 mg/kg b.wt) at 0 h, 48 h and 14 days after theadministration of Croton membranaceus aqueous root extract on Sprague–Dawley rats. [WBC = white blood cells; RBC = red blood cells; HGB = haemoglobin; HCT = haematocrit;MCV = mean corpuscular volume; MCH = mean corpuscular haemoglobin; MCHC = mean corpuscular haemoglobin concentration; PLT = platelet; LYM % = lymphocytes per-centage; LYM = lymphocyte count; RDW-SD = standard deviation in red cell distribution width; RDW-CV = coefficient of variation in red cell distribution width; PDW = plateletdistribution width; MPV = mean platelet volume; P-LCR = platelet larger cell ratio].
Fig. 4. �-Glutamyltranspeptidase levels post-extract administration. This figureshows that �-glutamyl transpeptidase was significantly raised when the 3000 mg/kgb.wt aqueous leaf extract was administered to S–D rats. Differences between the HDgtwt
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roup (3000 mg/kg b.wt) were significant compared to the C group (p < 0.05) (#). Fur-hermore, differences between the HD group and the LD group (1500 mg/kg b.wt)ere also significant (p < 0.05) (*). These differences occurred 48 h post administra-
ion but disappeared by day 14.
s AST and ALT did not show statistical differences.�-GT levels were almost the same for all groups at time 0 h. At
8 h, �-GT levels of the Control group and the LD group were nottatistically significant. However, �-GT levels of the HD group was.8 ± 0.4 U/L compared to the Control group value of 0.9 ± 0.2 U/LFig. 4). Differences were statistically significant (p < 0.05). ALP,nother hepatobiliary enzyme was not affected by LD/HD admin-stration of Croton membranaceus.
Total protein, albumin and globulin were not affected by LD/HDroton membranaceus administration throughout the experiment.ther biochemical analytes which did not show statistical differ-nces at 0 h, 48 h and 14 days between the Control, LD and HD, wererea, creatinine and lactate dehydrogenase.
From Table 1, the total WBC was slightly elevated after 48 h inll groups. This returned to baseline values. The inter-group differ-nces at 0 h, 48 h and 14 days were not significant. Haematologicalndices namely RBC count, haemoglobin level, haematocrit (HCT),
ean corpuscular volume (MCV), mean corpuscular haemoglobin
(MCH), mean corpuscular haemoglobin concentration (MCHC),did not show significant differences throughout. Platelet countshowed wide variations from 0 h through 48 h to 14 days (Table 1).However, differences at any of the time-points were not sta-tistically significant. Percentage lymphocyte was fairly constantthroughout, although lymphocyte counts [9.7–11.1 × 103/�l (C),8.2–9.3 × 103/�l (LD) and 6.6–9.8 × 103/�l (HD)], showed greatvariation. The differences however, were not statistically signif-icant (Table 1). The actual measurement of the width of theerythrocyte distribution curve (RDW-SD) was fairly constant at var-ious time-points. Values were however higher at 14 days comparedto the 48 h and 0 h. The relationship of actual measurement of thewidth of the erythrocyte distribution curve and the mean erythro-cyte size (RDW-CV%) did not show much variation. Mean plateletvolume (MPV), platelet large cell ratio (P-LCR) varied from 2.8 to8.9%. However, differences between groups and at various time-points were not statistically significant (Table 1). In conclusion allhaematological indices did not show statistical differences at 0 h,48 h and 14 days.
Finally, organs and tissues that were harvested and macroscop-ically examined were normal.
4. Discussion
The haematological indices after the aqueous root extractadministration did not show any abnormality. Differences after 48 hand 14 days were not statistically significant. Thus Croton mem-branaceus does not impact negatively on the hematopoietic systemas shown in Table 1. However, the biochemical picture was dif-ferent. The renal system assessed by urea and creatinine levels,were normal at 48 h and 14 days post drug administration. Thecardiac system however, had some cardiac enzyme levels lowered.While LDH which is more cardiac specific than CK remained nor-mal, CK was not. At 48 h, both total CK and CK-MB had statisticallysignificant lower levels than the controls either with LD or HD Cro-ton membranaceus (Figs. 1 and 2). Statistically, significant lowerlevels of CK-MB did not persist 14 days post drug administration(Fig. 2). Although, higher levels of these enzymes are of relevanceto myocardial infarction, low CK values are also seen in some clin-
ical conditions. This is partly seen when there is diminished effluxof the enzyme into the serum from diminished physical activity asa result of muscle wasting, advanced age or illness. Furthermore,reduced muscle mass as in alcoholics (Nanji and Blank, 1981) oraltered muscle permeability as reported in Cushing’s disease or
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42 G.A. Asare et al. / Journal of Ethn
atients on steroid therapy (Hinderks and Frolich, 1979) or indi-iduals on contraceptive (Fraser, 1980) may account for low CKevels. It has been reported that low CK values occur as a result ofhosphatase hydrolysis of phosphate substrates in the CK assay.dditionally, low CK activity is said to occur with macromolecularomplex formation as a result of enzyme-binding immunoglobu-ins causing steric hindrance to the enzyme. CK in the serum may beow because of reversible inactivation of the enzyme’s thiol groupshat may occur immediately after phlebotomy (Szasz et al., 1978).his may be the consequence of the release of the enzyme in aartly inactivated form or from CK inactivation within the circula-ion. Anti-hypertensive drugs have been found to lead to reducedK levels (Mueller, 1985). Low CK levels have also been associ-ted with low glutathione concentrations. It has been observed thatow circulatory CK associated with glutathione depletion cannot beverted by thiol-reducing compounds in the CK assay. Endogenouslutathione is thought to be a CK-preserving agent during periodshat the enzyme is present in circulation (Gunst et al., 1998).
Glutathione (GSH) is involved in a number of important cellularunctions, particularly the transport and storage of reduced GSH,rotection of cells against oxidative stress, detoxification of xeno-iotics and heavy metals, redox regulation of gene expression, androgression through the cell cycle (Meister and Anderson, 1983;ay et al., 1998; Noctor et al., 1998; den Boer and Murray, 2000).
ecause GSH participates directly in the cellular protection againstxidative stress, modification of the GSH metabolism by changinghe production of the enzymes involved in its regulation can be aseful approach to obtaining oxidative stress-tolerant plant modelsNoctor et al., 1998).
GSH conjugates are also substrates for �-GT. The enzyme playsrole both in detoxification of poisonous compounds (Ishikawa
t al., 1967; Meister, 1988) and the normal metabolism of biolog-cally active compounds such as leukotrienes and prostaglandinsCagen et al., 1976; Anderson et al., 1982; Meister, 1988; Ishikawa,992; Carter et al., 1997). �-GT is also an important enzyme thatodulates the redox status of thiols in the plasma membrane pro-
eins (Del Bello et al., 1999; Dominici et al., 1999) because one of theroducts of the �-GT reaction, Cys-Gly, contains a highly reactivehiol capable of producing active oxygen species by participating inhe Fenton reaction with iron ions (Halliwell and Gutteridge, 1989).
In certain plants, enzymes with �-GT activity are involved inecondary metabolism and they catalyze the synthesis of a rangef �-glutamyl dipeptides, which are formed during fruit ripeningnd accumulate in storage tissues such as seeds or bulbs [Blighiaapida (Kean and Hare, 1980), Glycine max (Ishikawa et al., 1967),sparagus officinalis (Kasai et al., 1982; Kawasaki et al., 1982)].
n onions, �-GT is said to catalyze the last step in the formationf volatile compound precursors by cleaving the �-glutamyl moi-ty off �-glutamyl alk(en) yl-Cys sulfoxides (Lancaster and Shaw,994). Whether plant �-GT participates in human GSH metabolism
s still an open question. It is not clear whether human GSH is arue substrate in vivo for the plant �-GT, because, in some cases,SH has been reported to be a poor in vitro substrate for thelant �-GT (Steinkamp and Rennenberg, 1984; Lancaster and Shaw,994). However, �-GT itself is a ubiquitous enzyme that catalyzeshe first step of glutathione (GSH) degradation in the �-glutamylycle in mammals. A cDNA encoding an rabidopsis homolog for-GT is over-expressed in tobacco (Nicotiana tabacum) plants. Aigh level of the membrane-bound �-GT activity is localized out-ide the cell in transgenic plants. The over-produced enzyme isharacterized by a high affinity for GSH and was cleaved post-
ranslationally in two unequal subunits. Thus, Arabidopsis �-GTs similar to that of the mammalian, in enzymatic properties,ost-translational processing, and cellular localization, suggest-
ng analogous biological functions of �-GT as a key enzyme inhe catabolism of GSH (Storozhenko et al., 2002). The high level
macology 134 (2011) 938–943
of �-GT observed in this study (Fig. 4) is more likely to be anover-expression of the enzyme as a result of exogenous supply,rather than the classical induction of the microsomal enzymes ofthe biliary tree. Serum �-GT measurement is principally used todiagnose and monitor hepatobiliary disease. Furthermore, �-GTpermits differentiation of liver diseases from other conditions inwhich serum ALP activity is elevated. Increased activities of bothenzymes in the serum with corresponding decrease in the liver sug-gest that the integrity of the membrane of the hepatocytes havebeen compromised as seen in other studies (Jeena et al., 1999;Clough et al., 2003; Brown et al., 2007). On the contrary, the rawdata of this study did not show significant difference in ALP lev-els at 0 h, 48 h and 14 days. Thus, the possibility of the elevated�-GT being attributable to hepatobiliary disease is unlikely. ASTand ALT levels were normal at any of the time points. However,conjugated bilirubin (CB) was higher in the HD group comparedto the C and LD groups at 48 h without a corresponding increasein �-GT in the HD group. This high CB was statistically signifi-cant but disappeared after 14 days. The hyperbilirubinemia did notcorrespond with any increase in AST and ALT as suggestive of hep-atic damage. The slight increase in CB in the HD group, thoughstatistically significant cannot be attributed to haemolytic jaun-dice either, as the haematological indices related to the RBC didnot show any abnormal results. Hepatic and hepatobiliary injuriescannot be substantiated and the histological micrographs showednormal liver.
In conclusion, Croton membranaceus ingestion does not produceacute toxicity. However, its CK lowering ability and possible exoge-nous �-GT supplementation could be explored in organ specificoxidative stress and oxidative damage.
Source of funding
Funding was provided by the School of Research and GraduateStudies, University of Ghana, Legon.
Acknowledgement
The authors disclose that the project was fully funded by theUniversity of Ghana Research Fund, through the School of Researchand Graduate Studies.
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