April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 3 (2) 219-224, <strong>April</strong> 2009. ISSN 0973-8916<br />
2. Materials <strong>and</strong> Methods<br />
2.1. Animals<br />
Wistar rats <strong>of</strong> either sex, weighing 100–<br />
150 g, were used. They were procured from the<br />
animal house <strong>of</strong> Mahaveera Enterprises (Reg.<br />
No.146/1999/CPCSEA), Ranga Reddy District,<br />
AP, India. They were housed in well-ventilated<br />
room at 27 0 C (±2) <strong>and</strong> photoperiod <strong>of</strong> 12-h light/<br />
dark cycle <strong>and</strong> fed with st<strong>and</strong>ard rodent pellet<br />
diet with tap water ad libitum. All procedures<br />
described were reviewed <strong>and</strong> approved by the<br />
Institutional animal ethical committee<br />
(Regn.No.169/1999/CPCSEA).<br />
2.2. Preparation <strong>of</strong> leaf extract<br />
The plant (B.roxburghii) growing in<br />
Karimnager Dist, Andhra pradesh, India was<br />
authenticated by Pr<strong>of</strong>. Raju S. Vastavaya,<br />
Taxonamist, Department <strong>of</strong> Botany, Kakatiya<br />
University, Warangal. Fresh leaves (Voucher<br />
number: LBR-055, deposited in: Herbarium,<br />
director: Pr<strong>of</strong>. Raju S.V.) from the plant were<br />
collected in the morning, the month <strong>of</strong> July 2008.<br />
Collected leaves were dried <strong>and</strong> powdered. The<br />
methanolic extract was prepared by maceration<br />
<strong>of</strong> leaves powder (1000g) with methanol (3L) for<br />
7 days with intermittent stirring. After extraction,<br />
the solvent was filtered <strong>and</strong> concentrated under<br />
reduced pressure. The extract (yield: 27%)<br />
obtained was stored at -20 o C until being used.<br />
2.3. Chemicals<br />
Silymarin was supplied as a gift sample<br />
by Micro Labs, Hosur, India. The solvents used<br />
were purchased from Merck India Ltd.<br />
(Mumbai). SGOT, SGPT, ALP <strong>and</strong> TBR<br />
estimation kits were purchased from Span<br />
diagnostics, Surat, India.<br />
2.4. Acute toxicity test<br />
The methanolic extract was administered<br />
orally only once in doses <strong>of</strong> 100, 300, 1000 <strong>and</strong><br />
2000 mg/kg to groups <strong>of</strong> mice (n = 6, age 35 days,<br />
220<br />
weight 18-22 g) <strong>and</strong> percentage mortality was<br />
noted beginning with 24 h up to a period <strong>of</strong> 7 days<br />
(13).<br />
2.5. Hepatoprotective activity<br />
Hepatic injury was induced in rats by<br />
subcutaneous administration <strong>of</strong> a single dose <strong>of</strong><br />
0.3 ml/kg CC1 4<br />
mixed with equal volume <strong>of</strong> olive<br />
oil on the 7th day, 2 h after the last treatment <strong>of</strong><br />
the drug (14). Animals were grouped as follows:<br />
GroupI: Control group, treated with<br />
2%w/v acacia in water at the dose <strong>of</strong> 2.0 ml, p.o<br />
(vehicle) daily for 7 days, followed by olive oil<br />
treatment (0.3 ml, s.c.) on day 7.<br />
Group II: Treated with vehicle (2.0 ml,<br />
p.o.) daily for 7 days followed by CCl 4<br />
on day 7.<br />
Group III: Treated with silymarin (100 mg p.o.)<br />
daily for 7 days followed by CC1 4<br />
on day 7.<br />
Groups IV <strong>and</strong> V: Treated with<br />
methanolic extract <strong>of</strong> BLR suspended in 5% gum<br />
acacia in water at doses <strong>of</strong> 200 <strong>and</strong> 400mg/kg<br />
daily for 7 days followed by CC1 4<br />
on day 7,<br />
respectively.<br />
2.6. Estimation <strong>of</strong> Biochemical Parameters<br />
The rats were sacrificed 24 h after the<br />
administration <strong>of</strong> last dose under anaesthesia<br />
using thiopentone sodium (35 g/kg b.w.i.p). The<br />
blood was collected <strong>and</strong> allowed to st<strong>and</strong> for 30<br />
min at room temperature <strong>and</strong> then centrifuged to<br />
separate the serum.The separated serum was<br />
estimated for various biochemical parameters like<br />
serum glutamate oxaloacetate transaminase<br />
(SGOT), serum glutamate pyruvate transaminase<br />
(SGPT) (15), Serum Alkaline Phosphatase (ALP)<br />
(16) <strong>and</strong> Total Bilirubin (17).<br />
2.7. Statistical analysis<br />
All values are expressed as means ± S.D.<br />
The data were subjected to one-way ANOVA<br />
Thirupathi et al