Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong> Vol. 3 (2) 219-224, <strong>April</strong> 2009. ISSN 0973-8916 2. Materials <strong>and</strong> Methods 2.1. Animals Wistar rats <strong>of</strong> either sex, weighing 100– 150 g, were used. They were procured from the animal house <strong>of</strong> Mahaveera Enterprises (Reg. No.146/1999/CPCSEA), Ranga Reddy District, AP, India. They were housed in well-ventilated room at 27 0 C (±2) <strong>and</strong> photoperiod <strong>of</strong> 12-h light/ dark cycle <strong>and</strong> fed with st<strong>and</strong>ard rodent pellet diet with tap water ad libitum. All procedures described were reviewed <strong>and</strong> approved by the Institutional animal ethical committee (Regn.No.169/1999/CPCSEA). 2.2. Preparation <strong>of</strong> leaf extract The plant (B.roxburghii) growing in Karimnager Dist, Andhra pradesh, India was authenticated by Pr<strong>of</strong>. Raju S. Vastavaya, Taxonamist, Department <strong>of</strong> Botany, Kakatiya University, Warangal. Fresh leaves (Voucher number: LBR-055, deposited in: Herbarium, director: Pr<strong>of</strong>. Raju S.V.) from the plant were collected in the morning, the month <strong>of</strong> July 2008. Collected leaves were dried <strong>and</strong> powdered. The methanolic extract was prepared by maceration <strong>of</strong> leaves powder (1000g) with methanol (3L) for 7 days with intermittent stirring. After extraction, the solvent was filtered <strong>and</strong> concentrated under reduced pressure. The extract (yield: 27%) obtained was stored at -20 o C until being used. 2.3. Chemicals Silymarin was supplied as a gift sample by Micro Labs, Hosur, India. The solvents used were purchased from Merck India Ltd. (Mumbai). SGOT, SGPT, ALP <strong>and</strong> TBR estimation kits were purchased from Span diagnostics, Surat, India. 2.4. Acute toxicity test The methanolic extract was administered orally only once in doses <strong>of</strong> 100, 300, 1000 <strong>and</strong> 2000 mg/kg to groups <strong>of</strong> mice (n = 6, age 35 days, 220 weight 18-22 g) <strong>and</strong> percentage mortality was noted beginning with 24 h up to a period <strong>of</strong> 7 days (13). 2.5. Hepatoprotective activity Hepatic injury was induced in rats by subcutaneous administration <strong>of</strong> a single dose <strong>of</strong> 0.3 ml/kg CC1 4 mixed with equal volume <strong>of</strong> olive oil on the 7th day, 2 h after the last treatment <strong>of</strong> the drug (14). Animals were grouped as follows: GroupI: Control group, treated with 2%w/v acacia in water at the dose <strong>of</strong> 2.0 ml, p.o (vehicle) daily for 7 days, followed by olive oil treatment (0.3 ml, s.c.) on day 7. Group II: Treated with vehicle (2.0 ml, p.o.) daily for 7 days followed by CCl 4 on day 7. Group III: Treated with silymarin (100 mg p.o.) daily for 7 days followed by CC1 4 on day 7. Groups IV <strong>and</strong> V: Treated with methanolic extract <strong>of</strong> BLR suspended in 5% gum acacia in water at doses <strong>of</strong> 200 <strong>and</strong> 400mg/kg daily for 7 days followed by CC1 4 on day 7, respectively. 2.6. Estimation <strong>of</strong> Biochemical Parameters The rats were sacrificed 24 h after the administration <strong>of</strong> last dose under anaesthesia using thiopentone sodium (35 g/kg b.w.i.p). The blood was collected <strong>and</strong> allowed to st<strong>and</strong> for 30 min at room temperature <strong>and</strong> then centrifuged to separate the serum.The separated serum was estimated for various biochemical parameters like serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) (15), Serum Alkaline Phosphatase (ALP) (16) <strong>and</strong> Total Bilirubin (17). 2.7. Statistical analysis All values are expressed as means ± S.D. The data were subjected to one-way ANOVA Thirupathi et al
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong> Vol. 3 (2) 219-224, <strong>April</strong> 2009. ISSN 0973-8916 followed by Newman-Keuls multiple comparison test <strong>and</strong> P
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