Analgesic and anti-inflammatory studies of the methanol

Extraction: The powdered plant material (500 g) was
extracted with methanol (2L) using soxhlets apparatus,
the solvent was removed in-vacuo to yield a residue
(56 g) subsequently referred to as Indigofera pulchra
extract .The extract was kept in a dessicator and a
fresh solution in distil water was prepared for each
experiment.
Chromatographic analyses of the methanol extract:
The chromatographic analysis was done by thin layer
chromatography (TLC) (silica gel on aluminium
plates).The methanol extract was spotted on to the TLC
plates and developed using three solvent system with
different polarities i.e. hexane/ethylacetate
(5:1),chloroform/methanol(19:1), and ethylacetate:
chloroform: methanol :water(15:8:4:1). The separated
components were visualized under visible and U.V.
light (254 and 366 nm) and sprayed with the following
reagents in order to reveal the spots of different
phytochemical groups: Dragendoff’s reagent for
alkaloids, 10% potassium hydroxide solution in
methanol for anthraquinones and coumarins ,
aluminium chloride for flavonoids and anisaldehyde
/sulphuric acid for steroid, terpenes, phenanthrenes and
dihydrostilbene nucleus [16] .
Animals: Adult Swiss albino mice of either sex
weighing between 19-22g and rats weighing 150-200g
obtained from the animal house facility, Department of
Pharmacology and Therapeutics, Ahmadu Bello
University, Zaria-Nigeria were used for these studies.
The animals were housed in a standard cage at room
temperature in a 12/12 hour light and dark cycle, and
were supplied with food and water ad libitum. All
experiments were conducted in accordance with animal
use ethics as accepted internationally.
Drug: Ketoprofen injection (Lek pharmaceuticals
company, Slovenia) and morphine (Martindale, Essex)
were used as standard drugs.
Route of Administration: The extract and the standard
drugs were administered to the animals intraperitoneally
in all the experiments.
Acute toxicity study: The study was divided into two
phases. In the first phase, nine mice of either sex
were divided into three groups of three mice each.
Group I received 10 mg/Kg extract while group II and
III received 100 and 1000 mg/kg extract respectively.
The mice were observed for signs and symptoms of
toxicity and mortality for twenty four hours after
treatment. In the second phase, 3 mice were divided
into 3 groups of one mouse each and treated based on
the result of the first phase. The first received extract
Res. J. Medicine & Med. Sci., 5(1): 106-110, 2010
107
at a dose of 1600 mg/kg, while the second and third
groups received the extract at doses of 2900 and 5000
mg/Kg respectively. The mice were also observed for
24 hours. The final LD 50 was calculated as the square
root of the product of the lowest lethal dose and the
highest non-lethal dose i.e. the geometric mean of
consecutive doses for which 0 and 100% survival rates
were recorded [17] .
Acetic acid – induced abdominal constrictions in
mice: Twenty five albino mice were divided into five
groups of five mice each. Group I was injected with
10 ml/Kg of normal saline (negative control). Group II
was injected with ketoprofen 10mg/kg (positive
control). Groups III, IV and V were injected with 50,
100 and 200 mg/Kg of the extract respectively. Thirty
minutes later, each mouse was injected with 10ml/kg
of aqueous solution of acetic acid (0.6%)
intraperitoneally. The number of abdominal
constrictions for each mouse was counted five minutes
after injection of acetic acid for a period of ten minutes
with a tally counter [18] . The percentage inhibition of
abdominal constrictions was calculated using the
following formula:
Inhibition (%) = Mean No. of writhes (Control) –
Mean No. of writhes (test) x 100
Mean No. of writhes (Control)
Hot plate test method: Mice were placed on a hot
plate maintained at temperature of 50±0.5 0 C. The time
taken for either paw licking or jumping (pain reaction
time) by each mouse was recorded. Mice that showed
initial nociceptive response within 20 seconds were
selected and used for the study. The mice were then
divided into 5 groups each containing 5 mice. Group
I served as negative control and received 10ml/Kg
normal saline while groups II, III and IV received the
extract (i.p) at doses of 50,100 and 200mg/Kg
respectively and the last group received morphine
(5mg/Kg). Thirty minutes later each mouse was placed
on a hot plate and the pain reaction time recorded [19] .
Carrageenan-induced Paw Oedema in Rats: Twenty
five mice were divided into 5 groups of 5 mice each.
Group I received 10ml/Kg normal saline group II
received ketoprofen 10mg/Kg (Positive control), while
groups III, IV and V received extract at doses of 50,
100 and 200 mg/Kg respectively. Thirty minutes later,
0.1ml of sterile saline suspension of 1% w/v
carrageenan was injected into the subplantar surface of
the left hind paw. Paw size was measured using vernier
caliper at time 0, 1, 2, 3, 4 and 5 hours after the
carrageenan administration [20] .