Nigerian Journal of Physiological Sciences, Vol. 19, No. 1-2, June/Dec, 2004, pp. 10-13

EFFECT OF SUB CHRONIC ADMINISTRATION OF ETHANOLIC LEAF EXTRACT OF CROTON ZAMBESICUS ON HEMATOLOGICAL PARAMETERS OF RATS

^*  J. E. OKOKON, K. C. IYADI, C. O. EFFIONG,

Department  of Pharmacology  & Toxicology, faculty  of pharmacy,  University  of Uyo, Uyo , Nigeria.

Received: June 21, 2004
Accepted: August 1^st , 2004

Code Number: np04003

SUMMARY

Ethanolic leaf extract of Croton zambesicus   was   administered  to rats at doses of 100 – 400mg / kg for 21 days to investigate its effect on the haematological indices of rats. Haematological indices, namely packed cell volume (PCV), Haemoglobin concentration (Hb) Red blood cell count (RBC), Mean cell Haemoglobin concentration (MCHC), Mean cell volume (MCV), and Mean   Corpuscular Haemoglobin (MCH) were assessed from whole blood obtained from the treated animals as well as those in the control group. The extract at the doses administered was found to  caused  reductions  in PCV, HB, RBC, MCH, MCH and WBC, in a  dose – dependent  fashion. However elevation of MCV was observed. This results indicate that the extract has  the  potential of  suppressing haemopoiesis  and causing anaemia.

Key Words:  Croton  zambesicus, haemotological indices, anaemia, rats.

INTRODUCTION

Croton zambesicus Muell Arg. (Euphorbiaceae) syn C.  amabilis  Muell. Arg.; syn. C. gratissimus Burch), is an ornamental tree grown in villages and towns  of Nigeria. It is a Guineo – Congolese species widely spread in tropical   Africa. The leaf decoction is used in Benin  as anti hypertensive  and antimicrobial  (urinary infections)( Adjanohoun et al, 2002). The Ibibios in Uruan  Area of AkwaIbomState use the leaf  traditionally   as malaria remedy. Block et al (2002) reported that ent – trachyloban – 3β  – ol, a trachylobane  diterpene, isolated from  dichloromethane  extract of  the leaves  has cytotoxic activity  on Hela cells. The alkaloidal fractions  of the leaf  have been reported to possess a weak activity against Klebsiella  pneumoniae, Pseudomonas aeruginosa,  Bacillis megaterium,  Bacillus  subtilis,   Eschericia  coli,  Proteus mirabilis,   Staphyloccocus    aureus, Aspergillus niger, Microsporum species and Penicillium  species (Abo et al  1999). The alkaloidal fractions of the stem have also been reported to be  active against all micro- organisms mentioned above (Abo et al 1999).  The essential oil found in the leaves contain P – cymene, linalool and beta – caryophyllene (Menut et al, 1995). The constituent of the essential oil also found in the flowering tops are pinene,  limonene, linalool, methol, carvone, thymol, alpha – humulene and  ceisnerolidol (Mekkawi, 1985). The study was aimed at investigating the effect of repeated administration of ethanolic leaf extract of Croton zambesicus on haematological indices namely packed cell volume, haemoglobin concentration,   red blood cell count, mean haemoglobin concentration, white blood  cell count and subsequently  to evaluate whether its  ethnopharmacological  uses  particularly as malaria remedy may have possible side effects such  as anaemia, which  is common with the  use of most chemotherapeutic  agents.

MATERIAL AND METHODS

Preparation of Plants Extract 

The leaves  of Croton Zambesicus Muell. Arg. (Euphorbiaceae) were collected August, 2003, from a compound in Uyo, AkwaIbomState, and authenticated by  Dr Uduak Eshiet, a taxonomist in the department of Botany, University of Uyo, Uyo Nigeria. Hebarium specimen was deposited at faculty of  pharmacy  hebarium  University  of Uyo, Uyo with voucher no. FPUU209.

The fresh leaves (2kg) of the plant were dried on a laboratory table for 8 days and reduced to powder. The powder 100g was macerated in ethanol (300ml) for 72 hours. The liquid extract obtained was concentrated in vacuo at 40°C.  The yield was 3.81%. The extract was stored in a refrigerator at 4°C until used for experiments reported in this study.

Animals

The animals used in the study were  young adult, male and females, (150- 180g) albino wistar rats obtained  from University of Uyo animal house, Uyo,  Nigeria. The animals were used after an acclimatization period of 10days to room temperature  and relative humidity  of 28+5°C and 50% respectively. The were housed in standard cages and maintained on standard animal pellets (Pfizer livestock feeds, AbaNigeria) and water ad libitum.

Experimental Design

The rats were weighed and randomly assigned on the basis of  weight into   5 groups of 5 animal each. Animals in Groups A, B, C and D were orally administered with 400, 300, 200 and 100 mg/kg of the extract respectively, while animals in group E were orally given normal saline 5 ml/kg and served as control.  Administration of the extract continued for 21 days between 0.8.00 and 0.9:00 hour each day. Twenty four hours  after the last administration, the  animals  were  anaesthetized with  chloroform vapour and dissected. Whole blood was obtained by cardiac puncture from each rat and collected into  anticoagalant – treated  (EDTA 0. 77M) sterile bottles. This was used for haematological studies. Blood haemoglobin (Hb) was determined spectrophotometrically by the  cyanomethaemoglobin method (Jain, 1986), packed cell volume (PCV) was determined  by method  of Jain, (1986).  Red Blood Cell (RBC) count was estimated by haemocytometer method of Arowolo (1982). Blood was diluted in 1,200 Dacie’s fluid which keeps and preserves the integrity of the RBC. For White Blood Cell (WBC) counts the dilution factor was 1:20 using 2-3% solution of acetic acid to which  gentian  violet was added.

The calculations for red cell indices were made as described by Jain (1986) Mean Corpuscular Volume (MCV) =  PCV x 10 / RBC (fl)

Mean Corpuscular  Haemoglobin (MCH) = Hb x 10 / RBC (pg)

Mean corpuscular Haemoglibin concentration = Hbc  x 100 / PCV

All data obtained were statistically analysed using students’ t-test. The data were expressed as average mean ± standard error and values of P <0.05 were considered significant.

RESULTS

The results of the effect of sub chronic administration of ethanolic leaf extract of Croton zambesicus on haematological indices of rats is presented in Table 1. Treatment of rats for 21days with the extract resulted in a dose- dependent reduction of PCV. The reduction was significant (P < 0.5) in group A with a value of 50.7 + 5.9% (mean +S.D) compared to control value of 56.7+4.8%. The values for group B, C and D were 52.5 + 7.6,53.2+4.9 and 55.3 + 2.5% respectively (Table 1). The blood haemoglobin concentrations (g/dl) were reduced in a dose – dependent fashion with significant  (P< 0.05) decrease recorded in groups A and B relative to control. The values were 12.3 + 0.8, 13. 2 + 1.0, 14.6 + 0.3, 15.5  + 0.8 and 16.0 + 0g/dl for groups A, B, C, D, and E respectively A non – significant  (P< 0.5) reduction in RBC counts of rats treated with the various doses  of the extract was  observed. The counts were 8.84+ 1.1, 9.7+0.5, 9.7+0.6, 10.27+2.0 millions per ml of blood respectively for groups  A, B, C and  D (Table 1), while the  control group had a count  of  10.6+ 0.63 millions per ml  of blood. There was a dose  - dependent decrease in the value of MCHC calculated. A significant  (P<0.05) decreased was recorded from rats in groups A with a value of 24.3 + 2.4% compared to control group with 28. 22 + 1.0%. The values were 25.14 + 1.2,  27. 4 + 0.9 and 28.02 + 3.1%  for groups B, C and D  respectively. The calculated values of MCH showed a decrease compared to control in a dose – dependent fashion, although the   differences were not  significant. For groups A, B, C, and D, MCH values of 13. 91+ 5.5, 13.6+2.0, 15.05+4.6 and 15.06 + 3.0% were recorded  respectively, while  that of control group was 15. 09 +1.0%. Elevation of MCV values was observed. A significant  (P<0.05) elevation was recorded in group A with a value of 57.35 + 0.6mm³ compared to control value of 53.49 + 0.4mm³. MCV values were 54.12+ 1.1, 54.84+0.7, and 53.74+1.0mm³ for groups B, C, and D respectively. The white blood cell counts (thousands /ml) for groups A, B, C, and D were 7.7 + 3.3, 9.0+0.8, 13.8+ 0.6 and 14.0+ 4.9 respectively and the control had a counts of 14.7 + 6. 0/ m l of blood. The count was observed to reduce with increased doses.

DISCUSSION

Administration of ethanolic leaf extract of Croton Zambesicus  to rats  for  21 days produced a dose – dependent reduction in PCV, Hb, RBC, MCHC, MCH and WBC. Elevation of MCV values of the treated rats was also recorded. The dose dependent reduction in PCV, Hb and RBC may have resulted from hemolytic activity of the extract leading to the observed reduction. It could also be a result of suppressive action of the extract on erythropoiesis. These reductions result in  anaemic condition. Keele et al (1983) reported that normocytic, hypochronic  anaemia results when there  is a reduction in  MCHC and MCH, while MCV as normal. A similar case was observed in this study though the MCV values are slightly elevated. Total white blood cells were observed to have reduced following the administration of the extract to rats. The decrease observed may have resulted from suppression of leucocytosis by the extract and also from the suppression of their production in the bone marrow. Further studies to confirm this as well as evaluate its mechanism of action are suggested. 

ACKNOWLEDGEMENT

The authors are grateful to Mr Nsikan Malachy of Dept. of Pharmacology and Toxicology, University of  Uyo for his  technical  assistance.

© Physiological Society of Nigeria 2004