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HEMATOLOGICAL PARAMETERS OF ALLOXAN-INDUCED DIABETIC

RATS TREATED WITH ETHANOL EXTRACTS AND FRACTIONS OF
NAUCLEA LAFILOIA LEAF

Article in European Scientific Journal · January 2013

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 European Scientific Journal September 2013 edition vol.9, No.27 ISSN: 1857 – 7881 (Print) e - ISSN 1857- 7431

HEMATOLOGICAL PARAMETERS OF
ALLOXAN-INDUCED DIABETIC RATS TREATED
WITH ETHANOL EXTRACTS AND FRACTIONS
OF NAUCLEA LAFILOIA LEAF

Asanga E. Edet, MSc

Ebong E. Patrick, PhD
Department of Biochemistry, University of Calabar, Nigeria
Eseyin, A. Olorunfemi, PhD
Department of Pharmaceutical and Medicinal Chemistry,
University of Uyo, Nigeria

Abstract
Hematological parameters which have been implicated in diabetes
mellitus were investigated in this study. N-hexane, ethyl acetate, butanol and
methanol fractions of the ethanolic leaf extract of Nauclea latifolia were
orally administered once daily for 2 weeks to diabetic rats. The levels of
RBC, Hb, HCT, MCV, MCH, MCHC, PLT, PCT, MPV, PDW, WBC,
lymphocyte and granulocyte were evaluated in blood. There was significant
reduction (P < 0.05) in RBC and HCT levels in the treatment groups of ethyl
acetate fraction (250 mg/kg) and ethanol extract (250 mg/kg) with significant
increases (P < 0.05) in their MCV and MCH levels when compared with the
diabetic control group. Significant increases (P < 0.05) in PLT levels of the
treatment groups of ethanol extracts, n-hexane fractions and ethyl acetate
fraction (100 mg/kg); PCT levels of ethanol extracts group and MPV levels
of ethyl acetate fractions treatment groups was high. The treatment groups of
glibenclamide, butanol, methanol, n-hexane, ethyl acetate fractions and
ethanol extract (250 mg/kg) showed significant reduction (P < 0.05) in their
WBC and lymphocyte levels while significant increase (P < 0.05) in
granulocyte levels was noted in the treatment group of ethanol extract (100
mg/kg) when compared with diabetic control group. In conclusion, the
ethanol extract proved to have anti-infective property. Some fractions,
showed capabilities to boost the immune system.

Keywords: Hematological parameters, Nauclea latifolia, diabetes

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Introduction
Diabetes mellitus is an endocrine disorder with different aetiologies,
it is characterized by aberration in carbohydrate, protein, blood relating
functions and fat metabolism caused by complete or relative insufficiency of
insulin secretion and action (Lebovitz, 1994; Andreoli et al., 1990).
Herbal medicine involves the use of herbs and plant parts (roots,
stems, leaves, barks, or even fruits to promote and improve health (Akinyemi
et al., 2005). Traditional medicinal practices on the African continent dates
as far back as 4000 years and were the sole medical systems for health care
before the advent of orthodox medicine (Eseyin et al., 2005). Plants are the
basis for the development of modern drugs and medicinal plants have been
used in many years in daily life to treat diseases all over the world (Agbor et
al., 2007).
Nauclea latifolium is a struggling shrub called pincushion tree. It is
grown in Africa and Asia (Okwori, et al., 2008). In Akwa Ibom State,
Nigeria, it is called Mbom-Ibong (Akpanabiatu et al., 2005) while the
Northern part of Nigeria name it Tabashiya (Gidado et al., 2008). Nauclea
latifolium leaves have been reported to have many medicinal potentials like
antidiabetic and hypoglycemic property (Gidado et al., 2008; Asanga et al.,
2012), hypolipidemic and hypocholesterolemic property (Asanga et al.,
2012), anti-hypertensive property (Nworgu et al., 2008). Some plants
extracts have been reported to destroy RBC thus leading to anemia (Adedapo
et al., 2007). Assessment of hematological parameters can be used to
determine the extent of deleterious effect of foreign compounds including
plant extracts and free radicals from alloxan on the blood constituents of an
animal (Mohammed et al., 2009). The aim of this study was to determine the
effects of ethanol extract and fractions of Nauclea latifolium leaves on the
hematological property of alloxan-induced diabetic wistar rats.
Materials And Methods
Fresh leaves of Nauclea latifolium were obtained from the endocrine
farm of Biochemistry Department, University of Calabar, Cross River State,
Nigeria. The leaves were identified in the department of Pharmacognosy,
University of Uyo, Akwa Ibom State, Nigeria. They were washed, dried
under shade, blended to powder (2kg). The powder was doubly macerated in
20 litres of 95% ethanol, filtered and concentrated in vacuo (40oC). The
concentrated ethanol extract (363.07g) was successively partitioned with n-
hexane (4 x 250ml), ethyl acetate (3 x 250ml), butanol (4 x 250ml) and
methanol (1 x 250ml) and all were concentrated in vacuo to obtain their
respective fractions (Venkateswarlu et al., 1993).
Animals/Diabetes Induction
Eighty albino (wistar) rats of weight range (100-250g) obtained from
animal house of department of Pharmacology and Toxicology, Faculty of

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Pharmacy, University of Uyo, Nigeria, were fed ad libitum with commercial

feed and clean drinking water and acclimatized for two weeks before the
experiment. They were fasted overnight and injected with alloxan
monohydrate (Sigma St. Louis, Mo, USA) intraperitoneally at a dose of 150
mg/kg body weight of rats as 5g/100ml distilled water. After 4 days, seventy
surviving rats with blood glucose levels above 250 mg/dL were considered
diabetic and used for the study (Katsumata et al., 1999; Dhandapani et al.,
2002 ).The experiment was conducted in compliance with ethical guide for
care and use of laboratory animals in the University of Uyo.
Treatment groups
The treatment groups used for the study were: Diabetic control
groups (30% Tween 80), glibenclamide (5mg/kg), ethanol extract (100
mg/kg), ethanol extract (250 mg/kg) n-hexane fraction (100 mg/kg), n-
hexane fraction (250 mg/kg), ethyl acetate (100 mg/kg), ethyl acetate (250
mg/kg), butanol fraction (100 mg/kg), butanol fraction (250 mg/kg),
methanol (100 mg/kg) and methanol (250 mg/kg). Each treatment group
consisted of five rats and the respective fraction and extract were
administered orally once daily via feeding tube for a period of two weeks
after which the animals were fasted overnight, anesthetized under chloroform
vapour, dissected, their blood collected through cardiac puncture and used
for the various hematology assay using the Mind Ray Automated
Haematology Analyser, Japan.
Statistical Analysis
The results in are presented as mean ± SEM at P < 0.05. The group
data were compared statistically using student t-test and one-way-ANOVA.

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Result And Discussion

Table 1: Effect of ethanolic leaf extract and fractions of Nuclea latifolia on hematological
parameters.

*= significant decrease, **= significant increases at p < 0.05

The results are as presented in Table 1. The primary reasons for

assessing the RBC is to check anemia and to evaluate normal erythropoiesis.
Hemoglobin level indicates the amount of intracellular iron, while
hematocrit, representing the volume of RBC in 100ml of blood helps to

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determine the degree of anemia or polycythaemia. The mean cell

hemoglobin level is a significant index for folic acid and or Vit B12 need
(Ganong, 1999). The resulting significant reduction (P < 0.05) in RBC levels
and HCT levels in the treatment groups of ethyl acetate fraction (250 mg/kg)
and ethanol extract (250 mg/kg) with significant increases (P < 0.05) in their
MCV and MCH levels when compared with the diabetic control group may
be due to hematotoxic effects associated with toxic substances on bone
marrow depression caused by damage to multiple classes of hematopoietic
cells and a variety of hematopoietic functions (Synder and Hadli, 1996).
Reactive O2 species generated during alloxan metabolism is implicated in red
cell damage (Rao et al., 2003), diabetic rats forms glycosylated hemoglobin
hence, decreased total hemoglobin (Sheela and Augusti, 1992). There was no
change (P < 0.05) in the red cell indices of the diabetic rats treated with
butanol, methanol, glibenclamide, n-hexane (250 mg/kg). This result was
similar to that reported by some researchers (Mohammed et al., 2009; Edet et
al., 2011).
Platelets are fragment of cells that participates in blood clotting, they
initiate repair of blood vessels walls and are also considered as an acute
phase reactant to infection or inflammation; plateletcrits showcases the
precise method of determining the degree of acute blood loss while mean
platelet volume (MPV) is used when investigating the ability of a drug to
enhance blood clotting (Ganong, 1999). The implication of the significant
increases (P < 0.05) in PLT levels in the treatment groups of ethanol extracts,
n-hexane fractions and ethyl acetate fraction (100 mg/kg) as well as in PCT
levels of ethanol extracts groups of rats and MPV levels in the treatment
groups of ethyl acetate fractions when compared with diabetic control group
is that they are potent as acute phase reactant to infection caused by alloxan
free radicals in the diabetic rats. This was consistent with the report
(Ajagbonna et al., 1999) on the ability of medicinal compounds or drugs in
altering the normal range of hematological parameters. More over, there was
no significant change in PLT levels of treatment groups of glibenclamide,
methanol, butanol fractions as well as in PCT levels of treatment groups of
glibenclamide, n-hexane, ethyl acetate, butanol and methanol fractions when
compared with the diabetic control group, suggesting that the plant fractions
may not cause thrombosis.
Alloxan diabetogenesis may cause perturbation in the bone marrow
stem cells (Edet et al., 2011). The significant reduction (P < 0.05) in WBC
and Lymphocytes levels of diabetic rats treated with methanol, butanol, n-
hexane, ethyl acetate fractions, glibenclamide and ethanol extract (250
mg/kg) when compared with the diabetic control group gave credence to the
abilities of the above treatment groups in curtailing hematological abuses in
the defense system of the diabetic rats. The result was not similar to that

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reported by some researchers (Mohammed et al., 2009; Akah et al., 2009;

Tanko et al., 2011). Also, the possible significant increase (P < 0.05) in
granulocytes levels of the rats treated with 100mg/kg of ethanol extract when
compared with the diabetic control group may indicate anti-infective effect
of the extract and this report was similar to the one by (Mohammed et al.,
2009; Tanko et al., 2011). More over, there was no change in granulocyte
levels noted for the treatment groups of methanol, n-hexane, butanol, ethyl
acetate fractions, glibenclamide and 250 mg/kg of ethanol extract. This
suggests their abilities in arresting allergy and bacterial infection resulting
from free radicals generated by alloxan metabolites on the different tissues of
the diabetic rats.
In conclusion, the ethanol extract proved to have anti-infective
property, glibenclamide, butanol, methanol, n-hexane, ethyl acetate fractions
and ethanol extract (250 mg/kg) showed capabilities to boost the immune
system and curtail some hematological abuse in the defense system, while
dose dependent ethanol extract and ethyl acetate fractions may reduce
erythropoiesis. Also, ethanol extracts, n-hexane fractions and ethyl acetate
fraction (100 mg/kg) may act as acute phase reactant to infections associated
with pathophysiology of diabetes mellitus.
It is hereby recommended that more work should be done on the
ethyl acetate, butanol and methanol fractions to isolate and identify the
components responsible for their hematoprotective property.

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