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Nigerian Journal of Pharmaceutical Sciences
Vol. 8, No. 1, March, 2009, ISSN: 0189-823X
All Rights Reserved
ANALGESIC AND ANTI-INFLAMMATORY ACTIVITIES OF THE METHANOLIC
LEAVES EXTRACT OF SECURINEGA VIROSA (EUPHORBIACEAE)
1*
M. Yerima,
1
M.G.Magaji,
2
A.H.Yaro,
3
Y.Tanko and
1
M.M. Mohammed

1
Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria
2
Department of Pharmacology, Faculty of Medicine, Bayero University, Kano, Nigeria
3
Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria

*Author for Correspondence: pharmyerima@yahoo.com, 234-8035960302

ABSTRACT
The methanolic leaf extract of Securinega virosa was evaluated for possible analgesic and anti-inflammatory activities in
rodents. Acetic acid induced writhing test in mice and formalin-induced pain in rats were used to study the analgesic
effect, while the effect of the extract on acute inflammation was investigated on carrageenan-induced paw oedema in
rats. The extract significantly (P <0.01) inhibited acetic acid induced writhing in mice and significantly (P <0.05)
attenuated the neurogenic phase of formalin-induced pain in rats at the highest dose tested. The extract also produced a
moderate anti-inflammatory activity which was found to be significant (P <0.05) at all the doses tested. Preliminary
phytochemical screening of the extract revealed the presence of alkaloids, tannins. saponins, cardiac glycosides,
flavonoids and resins. The intraperitoneal median lethal dose (LD
50
) of the extract in mice was found to be 1,265 mg/kg
suggesting that the extract may be relatively safe at the analgesic doses. The results obtained in this study lend credence
to the ethnomedical use of the plant in the management of pain and inflammatory conditions.Thus, supporting the
development of the biologically active substances as analgesics and anti-inflammatory agents.

Key words: Analgesic, Anti-inflammatory, Securinega virosa, Carrageenan, Formalin


INTRODUCTION
The long historical use of medicinal plants in
many traditional medical practices, including
experience passed from generation to
generation has demonstrated the safety and
efficacy of traditional medicine (Rukangira,
2001). However, scientific evaluation is
needed to provide evidences of their safety
and efficacy (WHO, 2000). Securinega virosa
is a low branching, dioecious shrub, or a small
tree, distributed throughout Tropical Africa
(Dalziel, 1936). The plant has enjoyed wide
patronage among the people of Tropical
Africa and its efficacy is widely acclaimed
(Neuwinger, 1996). The decoction of the
leaves and roots is used for abdominal pain in
Tanzania while the leave decoction is drunk
for fever by the Yorubas of South western
Nigeria. The decoction of the leaves with
other herbs is used in Northern Nigeria for
treatment of painful swellings. (Neuwinger,
1996).
Narcotic analgesics are associated with
addictive properties and numerous side effects
including respiratory depression, drowsiness,
decreased gastrointestinal motility, nausea and
alterations of endocrine and autonomic
nervous system activities (Almeida et al.,
2001). The search for pharmacological agents
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48

to overcome these shortcomings has become a
major goal in pain research. In view of this
and on account of the ethnomedical claim of
the usefulness of the plant in the management
of pain and inflammatory conditions, which to
our knowledge has not been scientifically
investigated, this present study was aimed at
investigating the analgesic and anti-
inflammatory activities of the methanolic leaf
extract of Securinega virosa in laboratory
animals.

MATERIALS AND METHODS
Collection Material
Fresh leaves of Securinega virosa were
collected from Basawa town, Sabon-Gari
Local Government Area of Kaduna State,
Nigeria in J uly, 2006. The plant was
authenticated at the Herbarium Section in the
Department of Biological Sciences, Ahmadu
Bello University (ABU), Zaria, Kaduna state,
Nigeria. A voucher specimen (No 918) was
deposited at the herbarium for future
reference.
Preparation of Extract
The leaves were air dried under shade for
twenty one days and then size-reduced into
powder with a pestle and mortar. About 100g
of the powdered leaves was macerated with
500ml methanol for 72hour with occasional
shaking. The extract was concentrated in
vacuo affording a yield of 13.2 %.w/w and
subsequently referred to as methanolic leaves
extract of Securinega virosa (SVMLE).
Solutions of the extract were prepared freshly
for each study.

Phytochemical Screening
The screening was carried out in accordance
with the standard protocol as described by
Trease and Evans (1983).

Experimental animals
Swiss albino mice of either sex (weighing 20-
30g) were obtained from the animal house
facilities of the Department of Pharmacology
and clinical Pharmacy, Ahmadu Bello
University, Zaria. The mice were maintained
on standard laboratory animal feed and water
ad libitum, and housed in polypropylene cages
at room temperature throughout the study.
These studies were carried out in Ahmadu
Bello University in accordance with the rules
governing the use of laboratory animals as
accepted internationally.

Drugs
The following chemicals and drugs were used:
carrageenan (Sigma-Aldrich), Acetic acid
(Ranbaxy Laboratories Ltd., Punjab),
Ketoprofen (Lek, Slovenia), Morphine
(Martinadale, Essex) and Piroxicam (Pfizer
laboratories, Pakistan). The methanolic leaves
extract of Securinega virosa (SVMLE) (25,
50, 100mg/kg).
Acute Toxicity study

The intraperitoneal LD
50
of the extract in mice
was conducted according to the method of
Lorke (1983). Briefly; the method was divided
into two phases. In the initial phase, 3 groups
of three mice each were treated with the
methanolic leaves extract of the plant at doses
of 10,100 and 1000mg/kg body weight i.p.
and observed for signs of toxicity and death
for 24 hours. In the second phase, 4 groups
each containing one mouse was injected with
four more specific doses of the extract based
on the result of the first phase. The LD
50
value
was determined by calculating the geometric
mean of the lowest dose that caused death and
the highest dose for which the animal survived
(0/1and 1/1).

Phytochemical screening of M. angolensis
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49

The phytochemical screening of M. angolensis
was carried out on the methanolic root bark
extract using standard protocol (Trease and
Evans, 1983).

Acetic acid induced writhing test in mice
Mice were divided into five groups each
containing 6 mice. The control group received
Normal saline (10ml/kg, i.p.). The test groups
were treated with 25, 50, 100 mg/kg/i.p. of
SVMLE while the fifth group received
piroxicam at the dose of 10mg/ kg, i.p. After
30 minutes of drug administration, the mice
were treated with 0.6% acetic acid at 10ml/kg
body weight, i.p. (Koster et al., 1959). Five
minutes after acetic acid injection, mice were
placed in individual cage and the number of
abdominal contractions was counted for each
mouse for a period of 10 minutes after 5
minutes latency, and the percentage inhibition
of writhing was calculated.

Formalin test in rats
The rats were divided into five groups each
containing 5 rats and were administered with
either normal saline (1ml/kg, i.p.), methanolic
leaves extract (25, 50 and 100 mg/kg, i.p) or
Morphine (4 mg/kg, s.c). Thirty minutes after
this treatment; 50 l of a freshly prepared
2.5% solution of formalin was injected
subcutaneously under the plantar surface of
the left hind paw of each rat. The rats were
placed individually in an observation chamber
and monitored for one hour. The severity of
pain response was recorded for each rat based
on the following scale: (0) rat walked or stood
firmly on the injected paw; (1) the injected
paw was favoured or partially elevated; (2) the
injected paw was clearly lifted off the floor;
(3) the rat licked, chewed or shook the
injected paw. Anti-nociceptive effect was
determined in two phases. The early phase
(phase 1) was recorded during the first
5minutes, while the late phase (phase 2) was
recorded during the last 45 minutes with a 10
minutes lag period in-between both phases.
(Dubuisson and Dennis, 1977; Tjlsen et al.,
1992).

Carrageenan-induced paw oedema
The rats were divided into five groups each
containing 5 rats. Acute inflammation was
induced by injecting 0.1 ml of (1%)
carrageenan into plantar surface of rat hind
paw (Winter et al., 1962). The methanolic leaf
extract (25, 50 and 100 mg/kg), normal saline
(1ml/kg) and ketoprofen (10 mg/kg) as
reference agent were administered 30 min
before carrageenan injection. The paw volume
was measured at 0, 1, 2, 3, 4 and 5h using a
vernier caliper to determine the diameter of
oedema. The difference between the readings
at time 0 h and different time interval was
taken as the thickness of oedema.

Statistical analysis
The results were expressed as Mean SEM
and analysed using Students t-test. A P value
<0.05 was considered significant.

RESULTS
The preliminary phytochemical screening of
SVMLE revealed the presence of flavonoids,
saponins, tannins, glycosides, alkaloids and
steroids (Table 1). The intraperitoneal median
lethal dose value of the extract in mice was
found to be 1,265mg/kg. The extract
significantly attenuated acetic acid induced
writhing in mice (P <0.001). The activity was
greater than that of piroxicam, the standard
agent, at the higher doses (Table 2). The
highest dose of the extract (100mg/kg)
significantly (P < 0.05) inhibited the first
phase of formalin-induced pain. Morphine, the
standard agent inhibited both phases (Table
3). The extract significantly (P < 0.05)
inhibited carrageenan-induced paw oedema at
the third hour. However, this was considerably
lower than that of ketoprofen which afforded
91.3% protection (Table 4).
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50

Table 1: Phytochemical screening of methanolic leaf extract of Securinaga virosa
Constituents Remark
Tannins +
Saponins +
Flavonoids +
Alkaloids +
Cardiac glycosides +
Cyanogenic glycosides +
Resins +
Steroid/Terpenoids +
Carbohydrates +
Anthraquinone -
Note: +Present, - Absent

Table 2: Effect of methanolic leaf extract of Securinaga virosa on acetic acid
induced writhing in mice
Treatment(mg/kg) Mean no.of writhes % inhibition
Normal saline 16.5 4.0
SVMLE 25mg/kg 5.3 2.2* 67.9
SVMLE 50mg/kg 2.8 0.9* 83.0
SVMLE 100mg/kg 3.0 2.1* 81.8
Piroxicam (10mg/kg) 4.8 1.7* 70.9
Each value represents mean SEM. *P <0.001 compared with control (Students t-test).
SVMLE=Methanolic leaf extract of S. virosa; n=6

Table 3: Effect of Methanolic leaf extract of Securinega virosa on Formalin-
induced pain in rats

Treatment(mg/kg)
Mean Pain Scores
First Phase Second Phase
Normal saline 3.0 0.0 3.0 0.0
SVMLE 25mg/kg 2.4 0.2* 3.0 0.0
SVMLE 50mg/kg 2.4 0.4 2.0 0.6
SVMLE 100mg/kg 1.4 0.7* 2.0 0.6
Morphine(5mg/kg) 1.2 0.4** 1.4 0.2**
Each value represents mean SEM. *P <0.05 **P <0.001 compared with control
(Students t-test) SVMLE=Methanolic leaf extract of S. virosa. n=5
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51

Table 4: Effect of Methanolic leaf extract of Securinaga virosa on Carrageenan-induced paw
oedema in rats
Treatment
Groups
Mean Paw Diameter (cm)
Time (h)
1 2 3 4 5
N/Saline
1ml/kg
0.19 0.01 0.25 0.03 0.23 0.02 0.20 0.03 0.20 0.02
SVMLE
25mg/kg
0.16 0.02
(15.8)
0.19 0.02
(24.0)
0.16 0.02*
(30.4)
0.13 0.02*
(35.0)
0.12 0.02*
(40.0)
SVMLE
50mg/kg
0.11 0.01**
(42.1)
0.16 0.03*
(36.0)
0.14 0.02*
(39.1)
0.120.02**
(40.0)
0.10 0.02*
(50.0)
SVMLE
100mg/kg
0.10 0.01**
(47.4)
0.15 0.01*
(40.0)
0.14 0.02*
(39.1)
0.09 0.02**
(55.0)
0.08 0.01**
(60.0)
Ketoprofen
10mg/kg
0.08 0.02***
(57.9)
0.07 0.03***
(72.0)
0.09 0.03***
(91.3)
0.10 0.02**
(50.0)
0.09 0.03**
(55.0)
Each value represents mean SEM. *P <0.05, **P <0.01 ***P <0.001, compared with control
values for corresponding hours. Figures in parentheses are percentage inhibition of inflammation.
SVMLE=Methanolic leaf extract of S. virosa. n=5

DISCUSSION
The acetic acid induced writhing test is very
sensitive and able to detect anti-nociceptive
effects of compounds at dose levels that may
appear inactive in other methods like tail flick
test (Collier et al., 1968; Bentley et al., 1981).
Increased level of prostanoids, particularly
PGE
2
and PGF
2a
(Derardt et al.,1980) as well
as lipoxygenase products (Levini et al.,1984;
Dhara et al.,2000) have been found in the
peritoneal fluid after intraperitoneal injection
of acetic acid. The analgesic effect of the
extract may therefore be due either to its
action on visceral receptors sensitive to acetic
acid, to the inhibition of the production of
algogenic substances or the inhibition at the
central level of the transmission of painful
messages. However, this model may not be
able to indicate the mechanism of analgesic
effect of the extract because other agents such
as antihistamines (Naik et al., 2000) and
myorelaxant (Koyama et al., 1997) are able to
reduce the pain induced by acetic acid.
Formalin test is a well established valid model
for the study of central sensitization events at
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52

the spinal level after peripheral inflammatory
state (Diaz and Dickeson, 1997). The two
distinct phases in formalin test are due to
direct effect of formalin on nociception and
due to inflammation with the release of
serotonin, histamine, bradykinin and
prostaglandins and at least to some degree, the
sensitization of central nociceptive neurons
(Dubuisson and Dennis, 1977; Huskaar and
Hole, 1987; Tjsen et al., 1992). Stimulation
of opioid receptors has also been suggested as
a possible mechanism of action against
neurogenic pain (Gaertner et al., 1999). The
ability of the extract to inhibit the second
phase of formalin-induced pain suggests that it
may possess central analgesic activity. The
probable mechanism of action of carrageenan-
induced inflammation is bi-phasic, the first
phase is attributed to the release of histamine,
serotonin and kinins in the first hour; while
the second phase is attributed to the release of
prostaglandins and lysosome enzymes in 2 to
3 hours (Brooks and Day, 1991). The extract
moderately inhibited the carrageenan-induced
inflammation in the 3
rd
hour. Analgesic and
anti-inflammatory effects have been observed
in flavonoids as well as tannins (Ahmadiani et
al., 2000). Flavonoids such as quercetin are
known to be effective in acute inflammation
(Rajnarayana et al., 2001). There are also
reports on the analgesics effects of alkaloids,
essential oils and saponins (Choi et al., 2005;
de Araujo et al., 2005; Reanmongkol et al.,
2005).
The analgesic and anti-inflammatory effect of
the extract may therefore, be due to the
presence of flavonoids, tannins, alkaloid or
saponins. However, further studies are in
progress in our laboratory to isolate the active
constituents responsible for the observed
effect, and to elucidate the possible
mechanisms of action responsible for the
analgesic and anti-inflammatory activities of
the methanolic leaf extract.

CONCLUSION
These findings suggest that the methanolic
leaf extract of Securinega virosa may contain
bioactive constituents with analgesic and anti-
inflammatory activities, and further support
the ethnomedical claim of the use of the plant
in the management of pain and inflammatory
conditions.
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