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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.
RESULTS Buncharoen et al. 4431 Table 1. Serum biochemistry in rats treated with 300 and 500 mg/kg body weight of S. aphylla root extracts for 45 days as compared to the control group. Biochemical index Control Treatment 300 (mg/kg body weight) 500 (mg/kg body weight) AST (IU/L) 136.67 ± 24.49 a 107.25 ± 18.12 a 115.50 ± 16.70 a ALT (IU/L) 28.83 ± 2.23 a 25.25 ± 2.63 a 23.00 ± 2.31 a ALP (IU/L) 96.83 ± 7.68 a 96.60 ± 16.55 a 92.25 ± 16.77 a BUN (mg/dl) 20.08 ± 2.38 a 19.46 ± 3.80 a 19.88 ± 2.68 a creatinine (mg/dl) 0.70 ± 0.03 a 0.64 ± 0.04 a 0.69 ± 0.04 a All values are expressed as mean ± SE. A same superscript letters within each row are not significantly different (P > 0.05). Table 2. Hematological values in rats treated with 300 and 500 mg/kg body weight of S. aphylla root extracts for 45 days as compared to the control group. Hematological parameter Control Treatment 300 mg/kg body weight 500 mg/kg body weight TRBC (cells/ml) 5.70 ± 1.90 a 6.95 ± 1.02 a 6.07 ± 1.22 a TWBC (cells/ml) 6.53 ± 2.85 a 5.33 ± 2.02 a 5.26 ± 2.10 a HGB (g/dl) 17.05 ± 3.66 a 18.57 ± 3.70 a 18.87 ± 4.09 a HCT (%) 48.51 ± 2.93 a 50.67 ± 3.01 a 52.25 ± 2.60 a MCV (fl) 95.21 ± 38.43 a 73.97 ± 9.45 a 88.94 ± 17.88 a MCH (pg) 32.07 ± 9.52 a 27.27 ± 7.31 a 32.21 ± 8.54 a MCHC (g/dl) 35.59 ± 9.35 a 37.04 ± 9.20 a 36.19 ± 7.44 a Lymphocyte (%) 76.71 ± 1.11 a 80.71 ± 1.38 b 82.43 ± 1.27 b Neutrophil (%) 16.38 ± 3.07 a 14.63 ± 2.13 a 13.88 ± 1.55 a Eosinophil (%) 1.13 ± 0.64 a 0.75 ± 0.46 a 0.88 ± 0.64 a Basophil (%) 1.00 ± 0.00 a 0.71 ± 0.49 a 0.86 ± 0.69 a Monocyte (%) 4.71 ± 2.36 a 2.86 ± 0.69 a 2.71 ± 0.76 a All values are expressed as mean ± SE. A different superscript letters within each row are significantly different (P < 0.05). Blood biochemical determinations The values of biochemical parameters of liver and kidney functions in this study are shown in Table 1. There were no significant differences (P > 0.05) in AST, ALT, ALP, BUN and creatinine of rats treated with S. aphylla extract at the doses of 300 and 500 mg/kg body weight when compared with those of the control group. All serum biochemical values were normal and within the reference ranges for rats. The reference ranges of AST, ALT, ALP, BUN and creatinine were 50 to 150, 10 to 40 and 30 to 130 IU/L, 12.0 to 25.8 and 0.4 to 2.3 mg/dl, respectively (Sharp and La Regina, 1998). Hematological studies The results of hematological parameters were listed in Table 2. There were no significant changes in TRBC, TWBC, HGB, HCT, MCV, MCH and MCHC of rats treated with S. aphylla extracts at the doses of 300 and 500 mg/kg body weight when compared with those of controls (P > 0.05). However, an increase in lymphocytes count was observed in all the treated groups. Histopathological studies Histopathological alterations were observed in liver tissues of rats treated with S. aphylla extracts at the doses of 300 and 500 mg/kg body weight. The sections of liver tissue in both treated groups showed similar damage, such as infiltration of leukocytes (Figure 1C) and haemorrhage in hepatic sinusoids (Figure 1D), whereas the normal features of liver tissue in the control group showed regular cords of hepatocytes with central vein and portal triad (Figure 1A and B). Moreover, the histological alteration was also observed in the kidneys of all treated groups. The renal tissues of the treated rats showed similarly significant changes, such as contracted
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B Journal of Medicinal Plants Resea
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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several studies. MO leaves ethanoli
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Journal of Medicinal Plants Researc
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Table 1. Pa and Pi of PTK inhibitor
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