April Journal-2009.p65 - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 3 (2) 219-224, April 2009. ISSN 0973-8916
2. Materials and Methods
2.1. Animals
Wistar rats of either sex, weighing 100–
150 g, were used. They were procured from the
animal house of Mahaveera Enterprises (Reg.
No.146/1999/CPCSEA), Ranga Reddy District,
AP, India. They were housed in well-ventilated
room at 27 0 C (±2) and photoperiod of 12-h light/
dark cycle and fed with standard rodent pellet
diet with tap water ad libitum. All procedures
described were reviewed and approved by the
Institutional animal ethical committee
(Regn.No.169/1999/CPCSEA).
2.2. Preparation of leaf extract
The plant (B.roxburghii) growing in
Karimnager Dist, Andhra pradesh, India was
authenticated by Prof. Raju S. Vastavaya,
Taxonamist, Department of Botany, Kakatiya
University, Warangal. Fresh leaves (Voucher
number: LBR-055, deposited in: Herbarium,
director: Prof. Raju S.V.) from the plant were
collected in the morning, the month of July 2008.
Collected leaves were dried and powdered. The
methanolic extract was prepared by maceration
of leaves powder (1000g) with methanol (3L) for
7 days with intermittent stirring. After extraction,
the solvent was filtered and 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 and 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 of 100, 300, 1000 and
2000 mg/kg to groups of mice (n = 6, age 35 days,
220
weight 18-22 g) and percentage mortality was
noted beginning with 24 h up to a period of 7 days
(13).
2.5. Hepatoprotective activity
Hepatic injury was induced in rats by
subcutaneous administration of a single dose of
0.3 ml/kg CC1 4
mixed with equal volume of olive
oil on the 7th day, 2 h after the last treatment of
the drug (14). Animals were grouped as follows:
GroupI: Control group, treated with
2%w/v acacia in water at the dose of 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 and V: Treated with
methanolic extract of BLR suspended in 5% gum
acacia in water at doses of 200 and 400mg/kg
daily for 7 days followed by CC1 4
on day 7,
respectively.
2.6. Estimation of Biochemical Parameters
The rats were sacrificed 24 h after the
administration of last dose under anaesthesia
using thiopentone sodium (35 g/kg b.w.i.p). The
blood was collected and allowed to stand for 30
min at room temperature and 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) and 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