p). Hesperidin powder was dissolved in 0.1% carboxy methyl cellulose and each rat received
daily 1 ml at a dose of 20, 40 and 80 mg/kg body weight orally by intragastric tube throughout the experimental period. The animals were randomly divided into six groups of six rats in each group. Group I: served as control (isotonic saline). Group II: animals were orally administered with hesperidin alone (80 mg/kg body weight). Group III: animals received ferrous sulfate (30 mg/kg body weight). Group IV-VI: animals were treated with ferrous sulfate (30 mg/kg body weight) following oral administration of hesperidin (20, 40, 80 mg/kg body weight) for 10 days. At the end of the experimental period, animals in different groups were sacrificed by cervical decapitation. Blood samples were collected without heparin for serum separation. Serum separated by centrifugation was used for various biochemical estimations. Rats were anesthetized by ketamine (28 mg/kg PI3K inhibitor body weight, intra muscularly) and the animals were sacrificed by cervical decapitation. The liver and kidney was quickly excised, rinsed with isotonic saline, blotted dry on filter paper, weighed and then 10% (w/v) homogenates of tissue was prepared in buffer (0.1 M Tris-HCL buffer (pH 7.4) and centrifuged at 3000 × g for 20 min at 4 °C. The resulting tissue homogenate was used for various biochemical assays. The activities of serum aspartate aminotransferase
(E.C.2.6.1.1), alanine aminotransferase (E.C.2.6.1.2), alkaline phosphatase (E.C.3.1.3.1) and lactate dehydrogenase (E.C.3.1.3.1) were assayed using commercially see more available diagnostic kits (Sigma diagnostics (I) Pvt. Ltd., Baroda, India). Gamma glutamyl transferase (E.C.2.3.2.2) activity was determined by the method of Rosalki et al., 1970 [16] using γ-glutamyl-p- nitroanilide as substrate. Based on Vanden Berg reaction, serum Phosphatidylethanolamine N-methyltransferase bilirubin was estimated by the method of Malloy and Evelyn, 1937 [17]. The activities of urea, creatinine and were estimated by Agappe Diagnostic (I) Pvt. Ltd., Kerala, India. Haemoglobin was estimated
by Drabkin and Austin, 1932 [18]. Creatinine clearance as an index of glomerular filtration rate was calculated from creatinine level in serum and creatinine level in 24 h urine sample. For determination of iron in blood, 1 ml of blood was digested with nitric acid in microwave oven. After digestion, iron was continuously pre concentrated and determined by flame atomic absorption spectrophotometry. A Perkin-Elmer 5000 atomic absorption spectrometer furnished with an iron hollow-cathode lamp (lamp current 4 mA) was used to determine the iron concentration. The instrument was set at 228.8 nm with a slit width of 0.5 nm. The acetylene flow rate was 2.0 l/min and an airflow rate of 17.0 l/min was employed to ensure an oxidizing flame. Lipids extracted from the tissues using by the method of Folch et al., 1957 [19].
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