J. Biomedical Science and Engineering, 2013, 6, 1040-1049 JBiSE
http://dx.doi.org/10.4236/jbise.2013.611130 Published Online November 2013 (http://www.scirp.org/journal/jbise/)
Some pharmacological studies on the methanolic extract of
Inula graveolense L.
Adnan J. M. Al-Fartosy
Department of Chemistry, College of Science, University of Basra, Basra, Iraq
Received 25 September 2013; revised 21 October 2013; accepted 31 October 2013
Copyright © 2013 Adnan J. M. Al-Fartosy. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Inula graveolens L. is widely used in Iraq for the
treatment of rheumatic fever, infant convulsions,
toothache, blood sugar, and also to dissolve internal
blood clots, and to aid digestion. However, the effi-
cacy and mechanisms of action of the plant remain
unclear. Therefore, the objective of the present study
was to investigate the pharmacological effects of the
methanolic extract (MEIG) of this plant belonging to
compositae family. Anti-diarrheal and antipyretic ac-
tivities of the extract were examined in rats. Anti-in-
flammatory and antinociceptive were studied in mice.
At the doses of 200 (P < 0.05) and 400 mg/kg body
weight (P < 0.01), the extract displayed remarkable an-
ti-diarrheal activity, evidence by a reduction in the
rate of defecation as well as by retardation of intesti-
nal transit of charcoal meal compared to normal sa-
line control group, dose dependently similar to lope-
ramide (5 mg/kg). The methanolic extract (400 mg/kg)
showed a significant (P < 0.01) dose dependent anti-
pyretic effect in yeast induced elevation of body tem-
perature in experimental rats. The methanolic extract
showed significant anti-inflammatory and antinoci-
ceptive activity at the dose of 400 mg/kg (P < 0.01) as
compared to standard drug diclofenac sodium (50
mg/kg). The extract inhibited paw and ear edema in a
dose-related manner. A dose-dependent analgesic ac-
tion was obtained against chemical (writhing test) and
thermal (hot-plate test) stimuli indicated that antino-
ciceptive activity may involve inhibition of pain by pe-
ripheral and central mechanisms. Again, the metha-
nolic extract (MEIG) was subjected for in vitro pro-
tein anti-denaturation using Bovine serum albumin
and anti-platelet aggregation of human blood activity.
It was observed that the extract showed greater per-
centage of inhibition of BSA (P < 0.01) at the highest
concentration (400 µg/ml). The extract also showed
potential platelet aggregation inhibitory activity in
adose-dependent manner. The maximum inhibition
was observed at the dose 400 µg/ml (P < 0.01) compar-
ed to standard drug commercial heparin (20 µg/ml).
Keywords: Pharmacological Activities; Antidiarrhae;
Antipyretic; Anti-Inflammatory; Antinociceptive;
Anti-Denaturation; Platelet Aggregation; Inula
graveolens L.
1. INTRODUCTION
Diarrhea is a gastrointestinal disorder, characterized by
an increase in stool frequency and change consistency
[1]. From a long time ago, plant kingdom played an im-
portant role for discovering new drug source. A number
of therapeutic drugs were isolated from plant species. For
the treatment of diarrhea, medicinal plants are a potential
source of antidiarrheal drugs [2]. Due to poor hygiene
practices and malnutrition, children in developing coun-
tries frequently suffer from various forms of infections
which present as fevers [3]. These fevers are often ac-
companied by aches and pain which all lead to morbidity
and mortality. Herbal medicines are often used as reme-
dies in these conditions since as a result of poverty or-
thodox medicines may be unaffordable [4]. It is known
that a large number of plant species contain various bio-
active compounds that may have health-beneficial prop-
erties, anti-inflammatory, anti-oxidant and antimicrobial
effects, and their preventive and therapeutic use is in-
creasing [5]. Pain is a sensorial modality and primarily
protective in nature, but often causes discomfort. It is the
most important symptom that brings the patient to physi-
cian. Analgesics relieve pain as a symptom, without af-
fecting its cause, currently available analgesic drugs such
as opiates and NSAIDS are not useful in all cases due to
their adverse effects [6]. Pain and inflammation are asso-
ciated with many pathophysiologies of various clinical

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1041
conditions like arthritis, cancer and vascular diseases. A
number of natural products are used in various traditional
medical systems to treat relief of symptoms from pain
and inflammation [7]. The main cellular components sus-
ceptible to damage by free radical and oxidative stress
are lipids (peroxidation of unsaturated fatty acids in cell
membrane), proteins (denaturation), carbohydrates and
nucleic acids [8]. Both inflammation and free radical da-
mage are inter-related aspects that influence each other.
As said above, proteins are susceptible to undergo dena-
turation by formation of free radical and the mechanism
of inflammation injury is attributed in part to release of
reactive oxygen species from activated neutrophil and
macrophages [9]. Plant extracts are an alternative to the
currently used anti-platelet agents, because they consti-
tute a rich source of bioactive chemicals [10]. Inula gra-
veolens L. is widely distributed in Mediterranean region
and Middle East to West Pakistan. In Iraq (Basrah and
lower Iraq), this plant is well known in Arabic and Eng-
lish system as “Shuwaser, Suawaid” and “Strong-smel-
lind Inula”, respectively [11]. Information gathered from
some herbalists (in Basra governorate, Iraq) that the plant
is useful to reduce rheumatic fever, infant convulsions,
toothache, blood sugar, and also to dissolve internal
blood clots, and to aid digestion. As there is no scientific
report on the biological activity of the plant, the present
investigation was done to evaluate the possible anti-dia-
rrheal, antipyretic, anti-inflammatory, antinociceptive, an-
ti-denaturation of protein and anti-platelet aggregation
activities of the methanolic extract of Inula graveolens
(MEIG). The findings from this work may add to the
overall value of the medicinal potential of the plant.
2. MATERIALS AND METHODS
2.1. Chemicals
All chemicals were purchased from Sigma-Aldrich Co.
(St. Louis, MO), and solvents were from E. Merck (Darm-
stadt, Germany). All of the reagents were prepared in
deionized distilled water to eliminate the contamination
of metal ions.
2.2. Plant Material and Extraction Procedure
Inula graveolens L. plant, used in this study, was collected
on october 2012 from Abu-Al-Khaseeb region (Southern
of Basra), Iraq. The plant was botanically authenticated
and voucher specimens 3897 were deposited in the Her-
barium of Basra (Iraq, Basra, College of Science, Uni-
versity of Basra). A quantity (100 g) of powdered plant
was extracted in a Soxhelet apparatus with 80% metha-
nol, for 24 h. The methanol extract was filtered and eva-
porated to dryness under reduced pressure in a rotary
evaporator to afford 9.47 g of dry extract.
2.3. Animals
Albino rat (150 - 200 g) of both sex were used to study
the anti-diarrhea and antipyretic activity. Healthy albino
mice of either sex (20 - 30 g) were used for anti-in-
flammatory and antinociceptive activity. The animals were
housed in polypropylene cages (five in cage) under a 12
h light/12h dark cycle in a controlled temperature room
(25 ± 2˚C). All the animals were acclimatized to the
laboratory conditions for a week before use. They had
free access to food and water. All studies were carried
out by using five groups of six animals (3 males and 3
females).
2.4. Antidiarrheal Study
2.4.1. Caster Oil-Induc e d Diar rhea Test
This test was carried out by using the method of [12].
The animals were fasted for 18 h prior to the test. Test,
standard samples and control were administered orally
60 min before received of castor oil at a dose of 1
ml/animal (p.o). Then animals were placed in cages lined
with adsorbent papers and observed for 4 h for the pres-
ence of diarrhea defined as watery (wet), unformed stool.
The control group result was considered as 100%. The
anti-diarrhea effect was expressed as the average percent
inhibition of defecation, which is calculated by the fol-
lowing equation:


% Inhibition1VtVc100
Where Vt and Vc represent mean number of defeca-
tion caused by castor oil in control and standard or test,
respectively. Control received normal saline (1 ml/kg,
p.o) and loperamide (5 mg/kg, p.o) was used as standard
drug.
2.4.2. Gastrointestinal M otility Test
This test was performed according to the method previ-
ously described using charcoal as a diet marker [13]. The
animals were fasted for 18 h prior to the test. Test, stan-
dard samples and control were administered orally 60
min before received of castor oil at a dose of 1 ml/animal
(p.o). After 60 min of drug administration, all animals
were received 1 ml of charcoal meal (10% charcoal in
5% gum acacia) orally. Sixty minute later, all animals
were sacrificed, and the intestine was removed without
stretching and placed lengthwise on moist filter paper.
The gastrointestinal motility was expressed as the ave-
rage percent retardation of intestinal transit, which is cal-
culated by the following equation:


% RetardationBA100
Where B and A represent the average distances (cm)
travelled by the charcoal meal from the pylorus to caecum

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1042
and total length (cm) of the small intestine, respectively.
Control received normal saline (1 ml/kg, p.o) and lop-
eramide (5 mg/kg, p.o) was used as standard drug.
2.5. Antipyretic Study
The procedure described by [14] was adopted for this
study. Fever was induced in the rats by injecting 20%
(w/v) suspention of Brewer’s yeast (Saccharomyces
cerevisiae) at a dosage of 1 ml/kg body weight subcu-
taneously. The rectal temperature of each rat was re-
corded by clinical thermometer before and after 18 h of
yeast adminstration. Rats that did not show a minimum
increase of 0.5˚C in temperature 18 h after yeast injection
were discarded. Rectal temperature of all the rats was
then recorded at the time of 1, 2 and 3 h after oral ad-
ministration of both test and standard drugs. The control
group result was considered as 100%. The antipyretic
effect was expressed as the average percent reduction in
rectal temperature, which is calculated by the following
equation:


n
% ReductionBCBA100
Where B and A represent mean number of rectal tem-
perature after and before 18 h of yeast adminstration re-
spectively, while Cn represent rectal temperature after 1,
2 and 3 h oral administration of drug. Control received
normal saline (1 ml/kg, p.o) and paracetamol (33 mg/kg,
p.o) was used as standard drug.
2.6. Anti-Inflammtory Study
2.6.1. Carra geenan-Induced P aw Edema Test
The test was determined according to the technique of
[15]. After 0.5 h, 0.1 ml of 1% (w/v) carrageenan sus-
pension was injected subcutaneously to the plantar sur-
face of the left hind paw. The paw volume was measured
using a plethysmometer (model 7140, Ugo Basile, Italy),
immediately and 0.5, 1, 1.5 and 2 h after drug treatment.
The anti-inflammatory effect is expressed as the average
percent inhibition of edema, which is calculated by the
following equation:


% Inhibition1VtVc100
Where Vt and Vc represent the increase in paw vol-
umes of mice treated with drug and control, respectively.
Control received normal saline (1 ml/kg, p.o) and di-
clofenac sodium (10 mg/kg, p.o) was used as standard
drug.
2.6.2. Xylene-Induced Ear Edema Test
A published method by [16] was adopted. After 0.5 h,
0.03 ml xylene was applied to the anterior and posterior
surfaces of the right ear. The left ear was considered as
control. Two hours after xylene application, mice were
killed and both ears were removed. Circular sections were
taken, using a cork borer with a diameter of 7 mm, and
weighed. The increase in weight caused by the irritant
was measured subtracting the weight of the untreated left
ear section from that of the treated right ear sections. The
anti-inflammatory effect is expressed as the average per-
cent inhibition of writhes, which is calculated by the fol-
lowing equation:


% Inhibition1VtVc100
Where Vt and Vc represent the average writhes in the
drug and control groups, respectively. Control received
normal saline (1 ml/kg, p.o) and diclofenac sodium (10
mg/kg, p.o) was used as standard drug.
2.7. Antinociceptive Study
2.7.1. Hot- Pl a te Test
The hot plate test was assessed according to the method
described by [16], with minor modification. The tem-
perature of a metal surface was maintained at 55˚C ±
0.5˚C. Latency to a discomfort reaction (jumping, with-
drawal or licking of the paws) was determined before
and after drug administration. A cut-off time was 15 sec,
to avoid damage of the paw. Reaction time and the type
of response were noted using a stopwatch. The latency
was recorded before and 0.5, 1, 1.5 and 2 h after oral
administration of both test and standard drugs. Average
reaction times were then calculated and the percentage
variation calculated using following relation:

% Inhibition
Before treatmentafter treatment1100


Control received normal saline (1 ml/kg, p.o) and di-
clofenac sodium (10 mg/kg, p.o) was used as standard
drug.
2.7.2. Writhin g Tes t
The test was performed as described by [7]. Test, stan-
dard samples and control were administered orally 30
min before intraperitoneal administration of 0.7% (v/v)
acetic acid (volume of injection 0.1 ml/10g body weight).
The mice were placed individually into glass beakers and
5 min were allowed to elapse. The number of stretching
or writhing was recorded for the next 10 min. A per-
centage reduction in the writhing number was considered
evidence for analgesia, which is calculated by the fol-
lowing equation:


% Inhibition1VtVc100
Where Vt and Vc represent the average number of
writhes in the drug and control groups, respectively.
Control received normal saline (1 ml/kg, p.o) and di-

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1043
clofenac sodium (10 mg/kg, p.o) was used as standard
drug.
pernatant which contained platelets (i.e. platelet rich pla-
sma (PRP)). The remaining blood was centrifuged at
1200 × g for 10 min to obtain another supernatant which
did not contained platelets (i.e. platelet poor plasma
(PPP)). The cuvettes were incubated at 37˚C for 5 min.
The platelet aggregation was initiated by adding 20 µl of
10 µM adenosine di-phosphate (ADP) to 1 ml of PRP.
The platelet aggregation was recorded for 5 min at 600
nm. The effect of different concentration (100, 200 and
400 µg/ml) of methanolic extract (MEIG) was studied by
incubation with PRP at 37˚C for 5 min before the addition
of ADP. The inhibition percentage of maximal aggrega-
tion was calculated using the following equation:
2.8. Anti-Denaturation Study
This test was carried out by using the method of [17]
with slight modification. Briefly, 0.2% w/v bovine serum
albumin (BSA) was prepared in Tris-buffer saline. pH
was adjusted at 6.8 using glacial acetic acid. Stock solu-
tion of 10,000 µg/ml of methanolic extract (MEIG) was
prepared by using methanol as a solvent. From this stock
solution 3 different concentrations of 100, 200 and 400
µg/ml were prepared by using methanol as a solvent. In
Eppendorf tube, 50 µL of the extract and 5 mL of 0.2%
(w/v) BSA solution and 50 µL of methanol were added.
Test tubes containing the sample mixture were heated at
72˚C for 5 minutes. After cooling for 10 min, the ab-
sorbance at 660 nm was measured. The experiment was
performed in triplicate. The inhibition percentage of pre-
cipitation (denaturation of the protein) was determined
on % basis relative to the control using the following
formula:


% Inhibition1VtVc100
Where Vt and Vc represent the maximal aggregation
of the drug and control groups, respectively. Control was
the platelet poor plasma (PPP) and commercial heparin
(20 µg/ml) was used as standard drug.
2.10. Statistical Analysis


% Inhibition1VtVc100 The data were expressed as mean values ± SEM and test-
ed with analysis of variance followed by Dunnett’s t-test.
P-values < 0.05, 0.01 were considered to be statistically
significant.
Where Vt and Vc represent the average denaturation
of the drug and control groups, respectively. Control was
50 µL of methanol and Ibuprofen (100 µg/ml) was used
as standard drug. 3. RESULTS AND DISCUSSION
3.1. Antidiarrheal Study
2.9. Anti-Platelet Aggregation Study
The results of this study are presented in Tabl es 1 and 2,
respectively. The results revealed that the extract at the
doses of 100, 200 and 400 mg/kg, produced a dose de-
pendent decrease in the number of faecal matters and de-
creased propulsion of charcoal meal of the gastrointesti-
nal tract passed by the animals in castor oil-induced
A published method by [18] was adopted. Blood was
taken collected from healthy human volunteers who have
not taken any medication two weeks prior to participa-
tion in the study. The blood was mixed with 3.8% (w/v)
sodium citrate solution in a ratio of 9:1 and centrifuged
at 260 × g for 15 min at 20˚C in order to obtain the su-
Table 1. Effect of the methanolic extract of Inula graveolens (MEIG) on castor oil-induced diarrhea in rats.
Groups Dose mg/kg No. of faecal dr opping s in 4 h % Inhibition
Group I Control 19.2 ± 1.21 -
Group II Standard 5.7 ± 0.64** 70.31
Group III 100 13.6 ± 0.73 29.16
Group IV 200 9.4 ± 0.45* 51.04
Group V 400 6.1 ± 0.87** 68.22
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.
Table 2. Effect of the methanolic extract of Inula graveolens (MEIG) on charcoal meal-stimulated gastrointestinal transit.
Groups Dose mg/kg Intestinal length (cm) Distance traveled by charcoal (cm) % Retardation
Group I Control 89.34 ± 1.31 76.61 ± 1.2 85.75
Group II Standard 84.25 ± 1.12 36.13 ± 1.08** 42.88
Group III 100 84.20 ± 1.26 52.31 ± 1.11 62.12
Group IV 200 86.18 ± 1.72 47.22 ± 1.49* 54.79
Group V 400 84.33 ± 1.34 39.15 ± 1.72** 46.42
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1044
diarrheal model, respectively. At doses (200 and 400
mg/kg) of the extract, a significant (P < 0.05 and P <
0.01) inhibitions (51.04% and 68.22%, respectively) of
characteristic diarrheal feces were observed. The effect
of the highest dose of the extract was similar to that of
the standard drug, loperamide (5 mg/kg). Diarrhea results
from an imbalance between the absorptive and secretory
mechanisms in the intestinal tract accompanied by hurry
resulting in an excess loss of fluid in the faeces. In some
diarrhea the secretory component predominates while
other diarrhea is characterized by hypermotility [19]. Cas-
tor oil is a triglyceride characterized by a high content of
the hydroxylated unsaturated fatty acid ricinoleic acid
[20]. After oral ingestion of castor oil, ricinoleic acid is
released by lipases in the lumen, and considerable amount
of ricinoleic acid are absorbed in the intestine [21]. Pres-
ence of ricinoleate in small intestine, the peristaltic activ-
ity of small intestine increases as a result of permeability
of Na+ and Cl− changed in the intestinal mucosa. Secre-
tion of endogenous prostaglandin is also stimulated by
ricinoleate [22]. Prostaglandins of the E series are con-
sidered to be good diarrheogenic agents in experimental
animals as well as in human beings. The inhibitors of
prostaglandins biosynthesis are therefore considered to
delay castor oil-induced diarrhea. Prostaglandins are as-
sociated with changes in the bowel that stimulate diar-
rhea. Recent study shows that the laxative effect of rici-
noleic acid present in castor oil is due to the induction of
contraction of intestinal smooth muscle which is medi-
ated by activation of EP3 receptors on intestinal smooth
muscle [23]. Many anti-diarrheal agents act by reducing
the gastrointestinal motility and/or the secretion. Inhibi-
tors of prostaglandin biosynthesis delay castor oil induc-
ed diarrhea [12]. Methanolic extract of Inula graveolens
(MEIG) exhibit significant anti-diarrheal activity. Earlier
reports suggest that anti-diarrheal properties of medicinal
plants might be ascribed to tannins, alkaloids, flavonoids,
saponin and steroids [24]. The methanolic extract of Inula
graveolens (MEIG) contain higher amount of both phe-
nolic and flavonoid compounds which may be responsi-
ble for its effect [25]. Flavonoids, present in plant extract,
are reported to inhibit release of autacoids and prostag-
landins, thereby may inhibit motility and secretion in-
duced by castor oil [26]. However, the components res-
ponsible for the anti-diarrheal activity of Inula graveolens
L. are currently unclear. Therefore, further work must be
carried out to isolate and identify these components.
3.2. Antipyretic Study
The effect of methanolic extract of Inula graveolens L. is
illustrated in Tab le 3 . It provoked dose dependent reduc-
tion in body temperature when studied at 100, 200 and
400 mg/kg, p.o. during various assessment times (1 - 3 h).
Maximum protection (82.14%) was observed after 3 h of
drug administration at 400 mg/kg (P < 0.01). Standard
drug, paracetamol produced 88.23% protection after 3 h
drug treatment 33 mg/kg (p.o). Fever has been recog-
nized as a major sign of diseased condition right from the
very beginning of human civilization. The febrile re-
sponse is coordinated by the central nervous system
through endocrine, neurological, immunological and be-
havioral mechanisms [27]. The initiation, manifestations
and regulation of the febrile response are dependent on
the pyrogenic and anti-pyrogenic properties of various
exogenous and endogenous substances. There is a gen-
eral consensus that fever is caused by a regular rise in
body temperature above normal daily, fluctuations origi-
nating in conjucation with an elevated thermoregulatory
set point [28,29]. The neurons of thermoregulatory sys-
tem center are sensitive not only to changes in blood
temperature but to cold and warm receptors located in
the skin and muscle and thus maintain an appropriate ba-
lance between the heat production and loss [30]. In rou-
tine practice, non-steroidal anti-inflammatory drugs
(NSAID) are commonly prescribed for the treatment of
different conditions. It has been suggested that pros-
taglandins inhibition are involved in the antipyretic mecha-
nisms of NSAID through cyclooxygenase pathway [31].
As a result, it could be speculated that the pharmacolo-
gically active components from this extract (MEIG) might
contain active principle(s) that demonstrated inhibitory
action on cyclooxygenase and thus, they produced anti-
pyretic activity by preventing the formation of prostag-
landins or by increasing the concentration of the body’s
Table 3. Effect of the methanolic extract of Inula graveolens (MEIG) on yeast-induced pyrexia in rats.
Rectal temperature (˚C) Rectal temperature after
administration of drug ( ˚C) % Reduction
Groups Dose mg/kg
Normal (A) 18 h after yeast
administration (B)1 h (C1) 2 h (C2) 3 h (C3) 1 h (C1) 2 h (C2)3 h (C3)
Group I Control 37.87 ± 0.14 38.60 ± 0.12 38.57 ± 0.1538.58 ± 0.1138.55 ± 0.12 - - -
Group II Standard 37.62 ± 0.16 38.30 ± 0.17 37.87 ± 0.14** 37.80 ± 0.12** 37.70 ± 0.16** 63.23 73.52 88.23
Group III 100 37.67 ± 0.19 38.22 ± 0.18 38.12 ± 0.1838.02 ± 0.1738.00 ± 0.13 18.18 36.36 40.00
Group IV 200 37.37 ± 0.13 38.35 ± 0.15 38.01 ± 0.1237.85 ± 0.16*37.70 ± 0.20* 34.69 51.02 66.32
Group V 400 37.17 ± 0.20 38.01 ± 0.18 37.62 ± 0.15** 37.47 ± 0.16** 37.32 ± 0.14** 46.42 64.28 82.14
N
= 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1045
own antipyretic component [28,30]. Flavonoids are known
to target prostaglandins which are involved in the pyre-
xia [32]. Hence the presence of flavonoids in the metha-
nolic extract of Inula graveolens (MEIG) may be contri-
butory to its antipyretic activity.
3.3. Anti-Inflammtory Study
The results of this study for carrageenan and xylene tests
are presented in Tables 4 and 5, respectively. The extract
(400 mg/kg) prevented the formation of edema induced
by carrageenan and thus showed significant anti-in-
flammatory activity (P < 0.01). The methanolic extract
(MEIG) reduced the edema induced by carrageenan by
29.46% after 3 h injection of noxious agent as compared
to the control group that received normal saline (1 ml/kg).
On the other hand, applied of xylene to the anterior and
posterior surfaces of the right ear in control animals, pro-
duced a local edema that increased progressively to reach
a maximal intensity 2 h after the applied of the phlogistic
agent. The extract showed a significant (P < 0.05 and P <
0.01) dose dependent reduction at the doses of 200 and
400 mg/kg, respectively as compared to the control group
that received normal saline (1 ml/kg). Diclofenac sodium
(10 mg/kg) also caused a significant inhibition of paw
and ear edema volumes, respectively. Inflammation is a
normal protective response to tissue injury and it involv-
es a complex array of enzyme activation, mediator re-
lease, fluid extravasations, cell migration, tissue break-
down and repair [33]. According to our findings, the me-
thanolic extract (MEIG) produced potential anti-inflam-
matory effect when assessed in both carrageenan and xy-
lene tests in experimental mice, as shown in Tables 4 and
5, respectively. The most widely used primary test to
screen new anti-inflammatory agents measure the ability
of acompound to reduce local edema induced in mice
paw by injection of an irritant agent [15]. Carrageenan
induced inflammation is a useful model to detect oral
action of anti-inflammatory agents [34]. The develop-
ment of oedema in the paw of the mice after the injection
of carrageenan is due to release of histamine, serotonin
and prostaglandin like substances [35]. The early phase
(0.5 - 1 h) of the carrageenan model is mainly mediated
by histamine, serotonin and increased synthesis of pros-
taglandin in the damaged tissue surroundings. The late
phase is sustained prostaglandin release and mediated by
bradykinin, leukotrienes, polymorph nuclear cells and
prostaglandins produced by tissue macrophages [36]. A
number of natural products are used in various traditio-
nal medical systems to treat relief of symptoms from
pain and inflammation. The significant ameliorative ac-
tivity of the methanolic extract (MEIG) and standard drug
observed in the present study may be due to inhibition of
the mediators of inflammation such as histamine, seroto-
nin and prostaglandin. The carrageenan assay is a good
method for the comparative bioassay of anti-inflamma-
tory. The xylene-induced ear edema method [16], has
been widely employed to assess the transudative, oxida-
tive and proliferative components of chronic inflamma-
tion.
3.4. Antinociceptive Study
The results of this study for acetic acid induced writhing
and hot plate tests are presented in Tables 6 and 7, re-
spectively. The methanolic extract (MEIG) at the doses
of 100, 200 and 400 mg/kg caused an inhibition on the
writhing response induced by acetic acid. The maximal
inhibition (43.75%) of the nociceptive response was
achieved at a dose of 400 mg/kg (P < 0.01). The extract
Table 4. Effect of the methanol extract of Inula graveolens (MEIG) on carrageenan-induced paw edema in mice.
Oedema diameter (cm) % Inhibition
Groups Dose mg/kg 0 h 0.5 h 1 h 1.5 h 2 h 0.5 h 1 h 1.5 h2 h
Group I Control 0.82 ± 0.01 0.88 ± 0.030.9 ± 0.03 0.96 ± 0.021.01 ± 0.01- - - -
Group II Standard 0.68 ± 0.03 0.65 ± 0.020.64 ± 0.020.61 ± 0.05** 0.59 ± 0.02** 41.59 36.46 28.8926.32
Group III 100 0.80 ± 0.08 0.76 ± 0.010.74 ± 0.060.71 ± 0.040.70 ± 0.03*30.7 26.05 17.7813.64
Group IV 200 0.75 ± 0.07 0.70 ± 0.030.69 ± 0.050.66 ± 0.01*0.62 ± 0.04*38.62 31.25 23.3420.46
Group V 400 0.64 ± 0.02 0.62 ± 0.040.59 ± 0.070.57 ± 0.02** 0.54 ± 0.03** 46.54 40.63 34.45 29.46
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.
Table 5. Effect of the methanolic extract of Inula graveolens (MEIG) on xylene-induced ear swelling in mice.
Groups Dose mg/kg Ear swelling (mg) % Protection
Group I Control 6.7 ± 0.51 -
Group II Standard 3.6 ± 0.62** 46.26
Group III 100 5.4 ± 0.45 19.40
Group IV 200 4.8 ± 0.52* 28.35
Group V 400 4.0 ± 0.6** 40.29
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1046
Table 6. Effect of the methanolic extract of Inula graveolens (MEIG) on latency to hot-plate test.
Mean latency(s) before and after drug administration % Inhibition
Groups Dose mg/kg 0 h 0.5 h 1 h 1.5 h 2 h 0.5 h 1 h 1.5 h2 h
Group I Control 1.78 ± 0.17 1.56 ± 0.26 1.45 ± 0.19 1.26 ± 0.22 1.07 ± 0.21 - - - -
Group II Standard 4.12 ± 0.33** 5.49 ± 0.1** 4.63 ± 0.64** 3.68 ± 0.62** 1.76 ± 0.08** 56.79 71.58 68.68 65.76
Group III 100 2.32 ± 0.62 2.61 ± 0.38 1.78 ± 0.72 1.43 ± 0.25 1.21 ± 0.07 23.27 40.22 18.53 11.88
Group IV 200 2.45 ± 0.51 1.97 ± 0.46 2.27 ± 0.54 1.86 ± 0.81 1.32 ± 0.20 27.34 20.81 36.1232.25
Group V 400 2.91 ± 0.47 3.17 ± 0.43** 4.35 ± 0.32** 2.24 ± 0.73** 1.97 ± 0.24** 38.83 50.78 66.66 43.75
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as compared to control.
Table 7. Effect of the methanolic extract of Inula graveolens
(MEIG) on acetic acid-induced writhing in mice.
Groups Dose mg/kg No. of writhing % Protection
Group I Control 36.2 ± 0.44 -
Group II Standard 6.4 ± 0.39** 82.32
Group III 100 26.7 ± 0.33* 26.24
Group IV 200 19.5 ± 1.2** 46.13
Group V 400 15.8 ± 1.02** 56.35
N = 6, values are mean ± SEM, *P<0.05, **P < 0.01, dunnet test as compared
to control.
showed a significant dose-dependent reduction in the
number of writhing with approximately 26.24%, 46.13%
and 56.35% of inhibition respectively. The oral dose of
methanolic extract at 400 mg/kg (P < 0.01) elicited a
significant analgesic activity as evidenced by increase in
latency time on comparison with negative control at the
end of 0.5, 1, 1.5 and 2 h. The increase in latency time
was found in a dose dependent manner. Acetic acid causes
an increase in the peritoneal fluid level of prostaglandins
(PGE2 & PGF2a) as well as lipooxygenase products, in-
volving in part peritoneal receptors and inflammatory
pain by inducing capillary [37]. Collier et al. [38] postu-
lated that acetic acid acts indirectly by inducing the re-
lease of endogenous mediators, which stimulate the no-
ciceptive neurons. The most important transmission path-
ways for inflammatory pain are that comprising periph-
eral polymodal nociceptors sensitive to protons, such as
acid sensitive ion channels and to algogen substances,
such as bradykinin and cytokines. Although the writhing
test has poor specificity (e.g., anticholinergic, tricyclic
antidepressants and antihistaminic and other agents show
activity in this test), it is a very sensitive method of
screening the antinociceptive of compounds [39]. The
hot-plate test is commonly used to assess narcotic anal-
gesia. Although the central and peripheral analgesics re-
spond by inhibiting the number of contractions provoked
by chemical pain stimuli, only the central analgesics in-
crease the time of response in the hot plate test [40].
These observations tend to suggest that the methanolic
extract of Inula graveolens (MEIG) may possess cen-
trally- and peripherally-mediated antinociceptive proper-
ties. The peripheral antinociceptive effect of the extract
may be mediated via inhibition of cyclooxygenases and/
or lipooxygenases (and other inflammatory mediators),
which its central antinociceptive action may be due its
possible action as partial agonist of adrenergic, seroto-
ninergic, cholinergic and dopaminergic recptors [41].
3.5. Anti-Denaturation Study
In the present investigation, the effect of methanolic ex-
tract (MEIG) was evaluated against heat induced dena-
turation of Bovine serum albumin (BSA). The results are
summarized in Table 8. The present findings exhibited a
concentration dependent inhibition of BSA denaturation
by the test extract throughout the concentration of 100,
200 and 400 µg/ml. The extract has shown a significant
(P < 0.01) anti-denaturation activity (62.16% and
75.67%) on BSA at 200 and 400 µg/ml, respectively.
Ibuprofen a standard drug showed the maximum inhibi-
tion 86.48% at the concentration 100 µg/ml. Protein de-
naturation is a process in which proteins loss their terti-
ary structure and secondary structure by application of
external stress or compound, such as strong acid or base,
a concentrated inorganic salt, an organic solvent or heat.
Denaturation of tissue proteins is one of the well docu-
mented causes of inflammatory and rheumatoid arthritis.
Production of auto-antigens in certain arthritic diseases
may be due to denaturation of proteins in vivo [42].
Agents that can prevent protein denaturation therefore,
would be worthwhile for anti-inflammatory drug devel-
opment. The mechanism of denaturation probably in-
volves alteration in electrostatic, hydrogen, hydrophobic
and disulphide bonding [43]. Several anti-inflammatory
drugs have been reported to show dose dependent ability
to inhibit thermally induced protein denaturation [44].
Bovine serum albumin (BSA) on denaturation expresses
antigens associated to Type III hypersensitive reaction,
related to diseases such as serum sickness, glomerulo-
nephritis, rheumatoid arthritis and systemic lupus erythe-
matosus [45]. From the result of the present study, it can
be stated that the methanolic extract (MEIG) is capable
of controlling the production of auto antigen and thereby
it inhibit the denaturation of proteins and its effect was
compared with the standard drug Ibuprofen. The plant
extract revealed to contain phenolic compounds, known
to produce inhibitory effect on protein denaturetion [44].

A. J. M. Al-Fartosy / J. Biomedical Science and Engineering 6 (2013) 1040-1049 1047
Table 8. Effect of the methanolic extract of Inula graveolens
(MEIG) on anti-denaturation of bovine serum albumin.
Groups Dose µg/ml Absorbance at 660 nm % Inhibition
Group I Control 0.37 ± 0.04 -
Group II Standard 0.05 ± 0.02** 86.48
Group III 100 0.23 ± 0.04 37.83
Group IV 200 0.14 ± 0.01** 62.16
Group V 400 0.09 ± 0.06** 75.67
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as com-
pared to control.
Literature survey also suggests that, the antidenaturation
property of BSA was due to the presence of two interest-
ing binding sites in the aromatic tyrosine rich and alipha-
tic threonine and lysine residue regions of the BSA [17].
They have also reported that therapeutic molecules could
be activating the tyrosine motif rich receptor dually with
threonine that regulates signal transduction biological
pathways for their overall biological action [46]. Further
studies are needed to elucidate other mechanisms of the
anti-denaturation activity of the methanolic extract (MEIG)
and to identify the active constituents responsible for the
anti-denaturation effect.
3.6. Anti-Platelet Aggregation Study
Platelets are essential for normal haemostasis. Activation
of the clotting cascade by trauma, results in platelet acti-
vation, which is followed by aggregation. Results of plate-
let aggregation were expressed as a percent of aggrega-
tion at a given time interval (5 min) from reagent addi-
tion (Table 9). The percentage aggregation inhibition of
the methanolic extract (MEIG) at 400 µg/ml and stan-
dard, commercial heparin at 20 µg/ml (P < 0.01) were
70.58% and 82.35% respectively. Platelets play an im-
portant role in the process of a thrombosis by adhering to
the damaged regions (caused by reactive oxygen species)
of the endothelial surface. The activated platelets to pla-
telets bond, binds also to leucocytes bringing them in to a
complex process of plague formation and growth. The
anti-platelet therapy constitutes the best available tool for
ameliorating the mechanism related to atherogenesis and
have interestingly inhibited platelet aggregation. Platelets
stick to the damaged vessel wall, they stick to each other
(aggregate) and release ADP, thromboxane A2 (TXA2)
which promotes further aggregation, and thus a platelet
plug is formed [47]. In the veins, due to sluggish blood
flow, the fibrinous tail is formed which traps RBC’s the
red tail. In arteries platelet mass are the main constituents
of the thrombus. Anti-platelet drugs are more useful in
arterial thrombosis, while anti-coagulant are more effec-
tive in venous thrombosis [48]. The methanolic extract
(MEIG) showed significant anti-platelet aggregation at
400 µg/ml. The mechanism behind this effect is yet not
Table 9. Effect of the methanolic extract of Inula graveolens
(MEIG) on platelet aggregation.
Groups Dose µg/mlAbsorba nce at 660 nm % Inhibition
Group I Control 0.34 ± 0.03 -
Group IIStandard 0.06 ± 0.01** 82.35
Group III100 0.24 ± 0.06 29.41
Group IV200 0.19 ± 0.01** 44.11
Group V400 0.10 ± 0.04** 70.58
N = 6, values are mean ± SEM, *P < 0.05, **P < 0.01, dunnet test as com-
pared to control.
clear but it might be said that the compound(s) respon-
sible for this effect are methanol-soluble, heat-resistant
plant botanicals, which might be different from chemical
anti-coagulating agents (salicylates). Natural compounds
were the first historical source of antithrombotic com-
pounds (heparin, vitamin K, antagonists, streptokinase and
urokinase). Recently several natural anti-platelet agents
from natural products including polyphenols [49] and fla-
vonoids [50] have been reported. Polyphenols may inhi-
bit platelet aggregation through a number of different me-
chanisms, including inhibition of cyclooxygenaes, lipoxy-
genase and phosphodiesterase activities [51]. Flavonoids
anti-aggregation effects may be attributed to inhibition of
thromboxane formation, thromboxane receptor antagoni-
sm, blunting hydrogen peroxide production, or inhibition
of phospholipase C [50,52].
4. CONCLUSION
From the results obtained in the present study, it may be
concluded that the methanolic extract of Inula graveolens L.
possessed good pharmacological activities, which might
be helpful in preventing or slowing the progress of va-
rious inflammatory, nociceptive, albumin denaturation
and plaetelet aggregation-related diseases and it showed
dose dependent activities. Further investgation on isol-
tion and identification of the active component(s) in the
plant may lead to chemical entities with potential for cli-
nical use.
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