Download Complete Issue (4740kb) - Academic Journals

4430 J. Med. Plants Res.
the users increasingly express an interest in the safety of
bioinsecticides. Therefore, information about the safety of
plants used as insecticides on mammals is still essential.
This study aimed to evaluate the effects of the ethanolic
extract from the root of S. aphylla on biochemical,
hematological and histopathological indices of male
albino rats. The data obtained from our study may be
used to assess the safety levels of this plant in mammals
and any benefit which may be received from the
development of bioinsecticide products.
MATERIALS AND METHODS
Plant
Fresh roots of S. aphylla were collected from Lampang province,
Thailand. The plant material was identified by the botanist from the
Department of Biology, Faculty of Science, Chiang Mai University.
A voucher specimen (number 09-111) was deposited at CMU
herbarium, Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai, Thailand.
Preparation of the plant extract
The roots of S. aphylla were washed with tap water, cut and dried in
an oven (50°C) to a consistent weight. The dried roots were soaked
in 95% ethanol for 3 days. The obtained extract was then filtered to
remove any residue, and then, it was evaporated by a vacuum
rotary evaporator to obtain the crude ethanolic extract. The
percentage yield of the extract was 7.92% (w/w). The ethanolic
extract was stored at 4°C until used. The residue was suspended in
distilled water, and required doses were prepared for future
experiments.
Animals
Male albino rats (Rattus norvegicus), 4 to 5 weeks of age, weighing
between 100 to 120 g, were purchased from the National
Laboratory Animal Center, Mahidol University, Salaya Campus,
Thailand.
The animals were housed in sanitary cages and had access to
tap water and a standard diet (C. P. 082). The room temperature
was controlled at 24 to 26°C in a 12 h light/dark cycle. All procedures
involving the animals were conducted with strict adherence
to guidelines and procedures reviewed and approved by the
Institutional Animal Care and Use Committee of the Biology
Department, Faculty of Sciences, Chiang Mai University.
Animal treatments
The rats were randomly divided into 3 groups (eight rats per group).
The animals in each group were given the root extracts of S.
aphylla orally at the doses of 300 and 500 mg/kg body weight daily
(1 ml/day) for 45 days. Control rats received only distilled water.
The selected doses in this study were based on the doses used in
chronic toxicity tests of this plant reported in a previous study
(Pandee et al., 2003).
Serum and tissues preparation
At the end of the treatment period, the animals were sacrificed and
blood samples were collected into non anti-coagulated and
ethylenediaminetetraacetic acid (EDTA) anti-coagulated tubes. The
non anti-coagulated blood was then centrifuged at 3,000 rpm for 10
min and serum was collected. Finally, the animals were quickly
dissected. Livers and kidneys were carefully removed. Portions of
these organs were fixed in Bouin’s solution for histopathological
examination.
Blood biochemical determinations
The level of clinical biochemistry such as aspartate aminotransferase
(AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), blood urea nitrogen (BUN) and creatinine were
evaluated to determine the function of the livers and kidneys of the
control groups and the treated rats.
All blood biochemical parameters were determined by automated
photometric systems with the cooperation of the Veterinary
Diagnostic Laboratory, Faculty of Veterinary Medicine, Chiang Mai
University.
Hematological studies
EDTA anti-coagulated blood samples were used to assay
hematological parameters. Total red blood cell counts (TRBC) were
determined by diluting blood samples with Grower’s solution, and
then red blood cell were counted in 5 red blood cells square of
hemocytometer. Total white blood cell counts (TWBC) were
counted in Neubauer’s hemocytometer after the blood samples
were diluted (1:20) in Turk’s solution. The hematocrit (HCT) was
measured by filling blood into a heparinized capillary tubes and
centrifuged at 11,000 rpm for 5 min. The HCT values were
measured with hematocrit reader.
Hemoglobin (HGB) concentration was done by adding 20 µl of
the blood sample into 5 ml of Drabkin’s solution. The optical density
was measured in a spectrophotometer at 540 nm and was
calculated from HGB standard curve. Some hematological parameters
such as mean cell volume (MCV), mean corpuscular
hemoglobin (MCH) and mean corpuscular hemoglobin concentration
(MCHC) were also calculated. For determining the
percentage of leukocytes, the blood smear was made and stained
with Wright’s dye. Finally, percentage of lymphocyte, monocyte,
neutrophil, eosinophil and basophil were counted under light
microscope.
Histopathological studies
The fixed tissues of rats were dehydrated with progressively
increasing concentrations of ethanol. The tissues were passed
through xylene solution to clear the ethanol and facilitate molten
paraffin wax infiltration (55°C).
After that, they were embedded in a wax block. Paraffin sections
of 6 µm thickness were cut with the rotary microtome and placed on
cleaned glass slides. Finally, the sections were stained with
hematoxylin and eosin. The stained slides were examined using a
light microscope where the photomicrographs of the tissue samples
were recorded.
Statistical analysis
For statistical analysis, data were analyzed by one-way analysis of
variance (ANOVA) using Statistical Package of Social Sciences
(SPSS) software version 16 for Windows. The results were
expressed as mean ± standard error (SE). A level of P value less
than 0.05 was considered to be significant.