ORIGINAL RESEARCH
Evaluation of Analgesic and Anti-Inammatory
Activities of 80% Methanol Root Extract of
Echinops kebericho M. (Asteraceae)
This article was published in the following Dove Press journal:
Journal of Inammation Research
Tesfaye Yimer
1
Eshetie Melese Birru
2
Meaza Adugna
2
Mestayet Geta
2
Yohannes Kelifa Emiru
3
1
Department of Pharmacy, College of
Health Science, Debre-Tabor University,
Debre Tabor, Ethiopia;
2
Department of
Pharmacology, School of Pharmacy,
College of Medicine and Health Sciences,
University of Gondar, Gondar, Ethiopia;
3
Department of Pharmacognosy, School
of Pharmacy, College of Medicine and
Health Sciences, University of Gondar,
Gondar, Ethiopia
Background: Pain and inammation are the major devastating health problems commonly
treated with traditional medicinal plants in Ethiopia. Echinops kebericho M. (Asteraceae) is the
one which is frequently used to treat pain and inammation by traditional healers in Ethiopian
folk medicine. However, the plant has not been scientically evaluated for its traditionally
claimed use. The present study aimed at the investigation of analgesic and anti-inammatory
activities of 80% methanol root extract of Echinops kebericho M. in mice model.
Methods: Successive maceration was used as a method of extraction using solvents of
increasing polarity: methanol and water. After extraction of the roots with 80% hydro
methanol, the crude extract was evaluated for its peripheral and central analgesic activities
using acetic acid-induced writhing test and hot plate method, respectively, while its anti-
inammatory activity was evaluated using carrageenan- and formalin-induced paw edema.
The extract was evaluated at 100, 200 and 400 mg/kg doses. The positive control groups
were treated with ASA 150 mg/kg for writhing test, morphine 10 mg/kg for hot plat method,
indomethacin 25 mg/kg and diclofenac 10 mg/kg for paw edema tests and vehicle, distilled
water (10 mL/kg) treated mice were assigned as negative controls. All treatment adminis-
trations were performed orally.
Results: E. kebericho extract at all test doses showed statistically signicant antinociceptive
activity in both chemicals-induced peripheral and thermal-induced central pain in a dose depen-
dent manner (p < 0.01 and p < 0.001). The greater analgesic activity was observed by the
maximum dose of the extract (400 mg/kg) in both acetic acids-induced writhing test (57.84%)
and hot plate method (69.40%). The effect of the extract was also statistically signicant (p <
0.01 and p < 0.001) in both carrageenan and formalin-induced paw edema in dose dependent
manner. Greater edema inhibition was observed by the highest dose (400 mg/kg) in both
observations with the respective percentage values of 70.00% and 79.87%, respectively.
Conclusion: In general, the data obtained from the present study elucidated that the extract
possessed a signicant analgesic and anti-inammatory activities and recommended for
further studies.
Keywords: analgesic activity, anti-inammatory activity, Echinops kebericho M.,
carrageenan, hot plate
Introduction
Pain can be dened as an unpleasant sensory and emotional experience associated
with actual or potential tissue damages, or described in terms of such damages.
1
It
is usually initiated by noxious stimuli and transmitted over specialized neuronal
networks to the CNS where, it is interpreted as such. It is a way to protect the body
Correspondence: Tesfaye Yimer
Tel +251 921313476
Email [email protected]
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from possible injury.
2
Similarly, inammation, which is
employed by both the innate and adaptive immune sys-
tems, is a protective response of the body to various
obnoxious stimuli such as infections and tissue injury.
3,4.
Regardless of the availability of sufcient drugs, pain
and inammation remain the most challenging and
devastating health problems which affect 80% of adult
population worldwide.
5
They are considered as the major
clinical, social, and economical problem in most com-
munities around the world.
6
Untreated and persistently
prolonged pain is the most pervasive disorder that results
both physical damage and psychological disorders.
7
Non-resolving inammation also leads to loss of func-
tion in terms of missing work, school, or social activities
and it results in the progression of serious inammatory
diseases, including asthma, autoimmune disease, chronic
inammation, glomerulonephritis, inammatory bowel
diseases, pelvic inammatory disease, reperfusion injury,
hypersensitivities, hay fever, atherosclerosis, and rheu-
matoid arthritis. These devastating conditions are the
major cause of disabilities and can lead to death unless
they are properly managed and controlled.
8
The currently available standard drugs for pain and
inammation remain the mainstay for managing and treat-
ing these disorders. However, they are associated with
many side effects and toxicities, such as gastric irritation,
gastric ulcer, alterations in renal function, effects on blood
pressure, hepatic injury, and platelet inhibition which may
result in increased bleeding. Using NSAIDs results in
increased risk of cardiovascular adverse events especially,
in patients taking COX-2 inhibitors.
8,9
Opioid analgesics
are also associated with many unwanted side effects and
toxicities, including drowsiness, nausea and vomiting,
pruritus, constipation, disturbing hormonal homeostasis,
hearing loss, tolerance, physical dependence, addiction,
and respiratory problems. In view of this, there is a need
for the intensication of research into medicinal plants
which are claimed to be effective in the management of
pain and inammation.
10
Traditional healers in different parts of Ethiopia use
a wide range of traditional medicinal plants to treatand
manage pain and inammation. But, the therapeutic poten-
tials of some of these medicinal plants have not been scien-
tically evaluated and validated so far.
11
Echinops kebericho
M. is among the traditional medicinal plants used in
Ethiopian folk medicine, and is frequently reported for its
antinociceptive and anti-inammatory potentials.
12,13
Traditionally, E. kebericho M has been used for the relief
of different infectious and non-infectious diseases including;
fever, headache, cough, diarrhea, malaria, as taenicide, sto-
mach ache, and typhus.
14
The infusion and inhalation (after
burning) of the roots is used to heal cough and headache
respectively, while inhalation of the leaf and steam after
burning is used to be relived from inammation which is
conventionally known as “mich” by traditional healers.
15
It is
reported that echinops species is used by traditional healers
for the treatment of a variety of conditions, such as; the dried
and/or fresh root is fumigated for the prevention of devil
sickness, the dried and/or fresh roots are crushed and mixed
with water and then a cup of the mixture is taken orally to
treat headache, as well as the fumigation of dried and/or fresh
roots are used for treating malaria.
16
The dried roots of
E. kebericho are crushed and macerated with fresh water
and mixed with coffee. The mixture is then taken orally to
relievetoothache, headache, and vomiting.
12
Traditional hea-
lers also use E. kebericho, by inhaling the dried roots to heal
inammation and evil eye.
13
Inhalation, infusion, and smok-
ing of the bulbs of E. kebericho is reported to be used for
treating cough and headache by traditional healers.
17
The species Echinops kebericho is found to possess
scientically approved pharmacological activities. Some of
them are as follows; 80% alcoholic root extract of the plant is
approved to have anti-schistosomal activity.
18
On extracts
the essential oils of the roots of E. kebericho is reported that,
they exhibited antimicrobial, anthelminthic, molluscicidal
and in vivo anti-plasmodial activities,
14
and 80% aqueous
extract of the roots of this plant is also reported to have
in vivo antidiarrheal and ex vivo spasmolytic activities.
19
Even though the plant E. kebericho M. is frequently
reported for its antinociceptive and anti-inammatory
potentials by traditional healers in different parts of
Ethiopia, no any scientic reports concerning the anti-
inammatory and analgesic activity of this plant have
been found in literatures yet. So, it is deemed prudent to
investigate the anti-inammatory and analgesic activities
of the plant scientically. The aim of the present study was
therefore, to investigate the analgesic and anti-
inammatory activities of 80% methanol root extract of
E. kebericho M. in a mice model.
Materials and Methods
Materials and Instruments
Rotary evaporator (yamato, Japan), lyophilizer (OPERON,
OPR-FDU-5012, Korea), digital plethysmometer (Ugo
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Basile-Cat no 7140, Italy) electronic balance (KERN-ALJ
220–4, Germany), tissue Drying Oven (Medite – Medizin
technik, Germany), syringes with needles, feeding tube,
hot Plate (Orchid Scientic, India.) were used with their
respective function.
Drugs and Chemicals
Carrageenan (Sigma Chemicals Co., St Louis, USA), for-
malin (Taen Industry, Ethiopia), normal saline
(H. R. Leuven, Belgium), distilled water (Ethiopian
Pharmaceutical Manufacturing Factory, Ethiopia), absolute
methanol (Indenta chemicals, India) and glacial acetic acid
(Sigma-Aldrich laborchemikalien, Germany), indometha-
cin (Cadila pharmaceuticals Ethiopia), aspirin, diclofenac
and morphine (Ethiopian Pharmaceutical Manufacturing
Factory, Ethiopia) obtained from the respective vendors
were used in the experiment.
Collection, Identication and Preparation
of Plant Materials
The roots of E. kebericho were collected from around Debre
tabor town South Gondar Zone of the Amhara regional state
which is located in North West direction and 667km far from
the capital city Addis Ababa. The plant was then authenti-
cated by Botanists in the Department of Biology, College of
Natural and Computational Sciences, University of Gondar
where a specimen with voucher number 002TYT/2019 was
deposited for further reference.
Preparation of the Extract
The roots of the plant were washed with tape water to
remove dust and any other debris present on it. The roots
of E. kebericho were then air dried under a shaded area at
room temperature and pulverized using a pestle and mortar
to get a coarse powder. A total of 1.50 kg powdered root
was macerated using 80% (v/v) methanol. The contents
were shaken manually each day and allowed to remain
within the solvent for 3 days. After 3 days, the extract was
ltered rst using gauze and then by Whatman lters
paper (No. 1). The marc was re-macerated twice using
the same volume of solvent to exhaustively extract the
plant material. After extraction was completed, the solvent
was evaporated under vacuum using rotary vapor and oven
at 40°C. The resulting solution was then placed in a deep
freezer operating at −20°C till it forms solid ice and then
the remaining solvent (water) was removed using lyophi-
lizer. After all a brownish gummy residue weighing
224gm of crude extract was obtained, giving rise to
a percentage yield of 14.93%. Then, the resulting crude
extract was kept within deep refrigerator (−20°c) till the
commencement of the main procedure.
Experimental Animals
Healthy adult Swiss Albino mice of either sex (20–35g,
and 6–8 weeks of age) were purchased from the Ethiopian
Health and Nutrition Research Institute (EHNRI) and
obtained from School of Pharmacy College of Health
Science University of Gondar. All mice were fed with
commercial pellets and have had access to water ad libi-
tum. The mice were acclimatized for a week before com-
mencement of the experiment in all procedures to
minimize stress. All mice used in this study were handled
in accordance with the internationally accepted standard
guidelines for use of laboratory animals.
20
Preliminary Phytochemical Screening
Standard phytochemical screening test was carried out to
detect the presence of secondary metabolites to relate the
analgesic and anti-inammatory of E. kebericho root
extract with the presence or absence of these active con-
stituents. Thus, the test for alkaloids, saponins, avonoids,
phenols, steroid, anthraquinone, glycosides and tannins
were performed using standard test procedures.
14,19.
Animal Grouping and Dosing
Swiss albino mice of either sex weighing 20–35 g were
randomly divided into 5 groups of 6 mice per group.
Group I was assigned as negative control and received
vehicles. Group II was served as positive control and
treated with standard drugs; morphine (10 mg/kg) for hot
plate test, indomethacin (25 mg/kg) for carrageenan test,
ASA (150 mg/kg) for writhing test and diclofenac (10 mg/
kg) for formalin test. Groups III–V were used as test
groups and given the extract of 100, 200 and 400 mg/kg
respectively. Doses were selected based on an acute toxi-
city study done previously.
14,20
All treatment administra-
tions were performed orally and the maximum volume
administered “was 0.015 mL/kg.”
Evaluation of Analgesic Activities of the
Extract
Acetic Acid-Induced Writhing Test
This method was conducted to detect the peripheral
analgesic activity of the extract and was performed by
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randomly dividing overnight fasted mice with free water
access as described in section (2.7). “Acetic acid (0.6%v/
v) (10 mL/kg, i.p)” was injected to all groups of mice
an hour just after the mice were given the extract, vehicle
or standard as per the respective groups. Analgesic activity
of the extract was assessed by counting the numbers of
writhing which consists of contraction of the abdominal
muscle together with stretching of the hind limbs for 30
min after a latency period of 5 min. A reduction in the
number of writhes as compared to the control group was
considered as evidence for analgesic potential of the
extract, and it was expressed as percent inhibition of wri-
thing as follows:
21
%analgesic activity ¼
mean writhing count
control treatedð Þ
mean writhing count
controlð Þ
� 100
Hot Plate Method
This method was conducted to evaluate the central analgesic
potential of E. kebericho extract and was performed by
introducing the mouse into an open-ended cylindrical space
with a oor consisting of a metallic plate that was heated by
a thermode. A plate was heated to a constant temperature of
55°C ± 1°C producing the behavioral components that were
measured in terms of their reaction times, namely paw lick-
ing withdrawal of the paw and jumping. All responses were
considered to be supraspinally integrated responses. Mice
were individually placed on a hot plate with a cut-off time
of 15 s to avoid lesions to the animals’ paws. The latency to
lick the paw or jump from the hot plate was recorded as the
reaction time. The reaction times were noted at 0 and 30, 60,
90 and 120 min after the administration of vehicle (distilled
water 10 mL/kg), standard drug (morphine10 mg/kg) and
100 mg/kg, 200 mg/kg and 400 mg/kg of the extract.
Percentage increase in reaction time or pain threshold inhibi-
tion, was calculated as follows:
22
%elongation ¼
latency testð Þ latency controlð Þ
latency testð Þ
� 100
Anti-Inammatory Activity
Carrageenan-Induced Paw Edema
This method was carried out by inducing of acute inam-
mation in the paws of overnight fasted mice with free
access of water. The mice were injected with carrageenan
(1% w/v in normal saline, 0.05mL) into the plantar side of
the left hind paw. Just before induction of inammation,
the leg of each mouse was marked on the skin over the
lateral malleolus, so that it could be immersed to the same
extent in the measurement chamber of the plethysmometer.
Carrageenan was injected one hour after administration of
the extract, the vehicle and the standard drug with the
respective groups of mice. Inammation was quantitated
in terms of mL i.e., displacement of water by edema using
a digital plethysmometer at time 0, 1, 2, 3, 4 after carra-
geenan injection. The percentage inhibition of edema was
calculated in comparison to the control mice as follows;
23
%Edema inhibition ¼
PEC PET
PEC
� 100
Where; PEC paw edema in control group
PET paw edema in test group
Formalin-Induced Pedal Edema
In this procedure, 2% v/v formalin was used for the induction
of sub-acute inammation. Freshly prepared 2% v/v formalin
with “distilled water (0.02mL)” was injected into the right
hind paw of overnight fasted mice by sub-plantar injection at
the 1
st
and 3
rd
days of observations. The right hind paw of
each mouse was marked at the level of lateral malleolus
before formalin induction so that it could always be
immersed to the same extent in the measurement chamber
of the plethysmometer throughout the observation days. The
extract, the standard drug and the vehicle were given as per
their respective grouping 1hr prior to formalin injection for 7
consecutive days. The mice paw volume was recorded daily
using Plethysmometer after 1hr of extract, drug and vehicle
administration till the 7th day and the percentage of edema
inhibition was calculated using the above formula.
24
Statistical Analysis
Analysis of results was done using Statistical Package for
Social Sciences (SPSS) software version 21. All results
obtained were expressed as mean ± standard error of mean
(SEM) of responses. The statistical signicance was deter-
mined by using One-way Analysis of Variance (ANOVA)
followed by a Tukey post hoc test to compare variations
among groups and the results were considered signicant
at p < 0.05. The analyzed data were then presented using
tables and graphs where necessary.
Results
Preliminary Phytochemical Screening
Preliminary phytochemical screening for secondary meta-
bolites was carried out to detect the presence or absence of
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different phytoconstituents from 80% methanol root
extract of E. kebericho. The presence of saponins, alka-
loids, phenols, tannins, avonoids, glycoside, and steroids
were conrmed through qualitative color changes of test
reagents which will give a clue to the possible mechanisms
of analgesic and anti-inammatory effects of the extract.
Analgesic Activity
Acetic Acid-Induced Writhing Test
In this test, E. kebericho extract at all test doses employed
(100, 200, and 400m g/kg) showed statistically signicant
peripheral analgesic activity in a dose dependent manner (p
< 0.05, p < 0.01 and p < 0.001 respectively) as compared to
the negative control. All 3 test doses of the extract produced
increased inhibition of the numbers of writhing with max-
imum inhibition observed at maximum dose (400 mg/kg)
(p< 0.001). The highest dose signicantly decreased the
number of writhing (p < 0.001) than the lower dose (100
mk/kg) and the middle dose (200 mk/kg) (p < 0.05). The
extent of reduction of writhing in the different doses of the
extract was different, i.e., signicantly lower in 100 mg/kg
(p < 0.001) and 200 mg/kg (p < 0.01) and comparable with
400 mg/kg as compared to the standard drug of 150 mg/kg
ASA (Table 1).
Inter group comparison among the three doses employed
also showed statistically signicant difference in 100 mg
versus 200 mg/kg (p < 0.05), 200 mg versus 400 mg (p <
0.05) and 100 mg versus 400 mg (p < 0.001). The maximum
dose of EK extract (400 mg/kg) showed comparable %
inhibition of the numbers of writhing with the standard
drug (ASA 150 mg/kg) with respective percentage values
of 57.84% and 60.98% respectively (Table 1).
Hot Plate Method
The hot plate method was performed to determine the central
analgesic activity of EK extract in mice. In this model all the
three test doses of EK extract and the standard drug morphine
produced signicant central analgesic activity (p < 0.05, p <
0.01 and p < 0.001) by delaying the reaction time at all-time
intervals of observation when compared with the negative
control (Table 2). The maximum analgesic activities of all
doses of the extract (100, 200 and 400 mg/kg) was observed
at 120 mins of observation with the respective values of
63.93%, 66.94%, and 69.40% as compared with the standard
drug (morphine 10 mg/kg) that produced 70.81% analgesic
activity (Figure 1).
The latency delayed by the lower and middle doses of
the extract was signicantly lower (p < 0.001) than that of
the standard drug, while the reaction time increased by the
higher dose was comparable with the standard drug at all-
time intervals of observation (Figure 1).
Anti-Inammatory Activity
Carrageenan-Induced Paw Edema
This model was conducted to evaluate the anti-inammatory
potential of the extract in acute phase of inammation. Sub
plantar injection of 0.05 mL of 1% carrageenan to the mice
hind paw produced a progressive increment of paw thick-
ness that reached its maximum value after 3hrs of induction
in negative control (Table 3). All test doses of EK extract
(100, 200, and 400 mg/kg) produced statistically signicant
inhibition of paw thickness starting from 1 hr (p < 0.01 and
0.001) and the effects persisted till the fourth hour of obser-
vation (p < 0.01 and p < 0.001) post carrageenan induction
as compared to the negative control.
The maximum percent of anti-inammatory activity of
the extract at all test doses was observed at the 4
th
time
with the respective percentage values of 54.62%, 61.54%
and 70.00% and the effect at this time was found to
increase in dose dependent manner. The maximum dose
(400 mg/k) of the extract produced higher paw edema
inhibition than the lower dose (100 mg/kg) and middle
dose (200 mg/kg) of the extract (p < 0.01 and p < 0.05
respectively), whereas its edema inhibition effect was
comparable with the positive control (indomethacin
25 mg/kg) at the 4
th
time of observation (Table 3).
Formalin-Induced Paw Edema Model
Formaldehyde-induced pedal edema method in mice was con-
ducted to detect the anti-inammatory potential of 80% hydro
methanol root extract of EK in sub-acute phase of
Table 1 Effect of 80% Hydromethanolic Root Extracts of
Echinops kebericho on Acetic Acid-Induced Writhing in Mice
Groups Mean No. of Writhing ± S.E.M %
Inhibition
DW 10 mL/kg 47.83 ±0.83a
3
c
3
d
3
e
3
——–
ASA 150 mg/kg 18.67 ± 0.42b
3
c
3
60.98
EK100 mg/kg 33.00 ± 1.48a
3
b
3
d
3
e
3
31.01
EK200 mg/kg 24.67 ±1.00a
1
b
3
c
1
48.43
EK400 mg/kg 20.17±1.42b
3
c
3
57.84
Notes: Analysis was performed with One-Way ANOVA followed by a Tukey post
hoc multiple comparison test. Data was expressed in mean ± SEM. (N = 6). a = as
compared to +ve control, b = as compared to -ve control, c = as compared to
100 mg/kg EK, d = as compared to 200 mg/kg EK, e = as compared to 400 mg/kg EK,
3p < 0.001, 2p < 0.01, 1p < 0.05.
Abbreviations: ASA, acetyl salicylic acid (150 mg/kg); DW, distilled water (10mL/
kg); EK100 mg, Echinops kebericho extract (100 mg/kg); EK200 mg, Echinops kebericho
extract (200 mg/kg); EK400, Echinops kebericho extract (400 mg/kg).
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inammation. Sub planar injection of 0.02mL of 2% v/v
formaldehyde with distilled water to the mice hind paw at
the 1
st
and 3
rd
day of observation produced progressive incre-
ment of the paw volume of the mice which were treated with
the negative control (distilled water 10 mL/kg) (Table 4).
E. kebericho extract at all test doses (100, 200 and 400 mg/
kg) signicantly decreased (p<0.05, p < 0.01, p < 0.001
respectively) paw edema volume in mice starting from the
1
st
day of formalin induction and then continued to the 7
th
day
(p < 0.01, p < 0.001) post-formalin induction as compared
with the negative control (Table 4).
The maximum percent of edema inhibition in all test
doses of the extract was observed at the 7
th
day of obser-
vation with the respective values of 69.46%, 74.83%, and
79.87% (Figure 2) and the effect at this time was found to
be dose dependent. Inter group comparison among the
different doses of the extract showed statistically signi-
cant different effect in 100 mg/kg versus 400 mg/kg at the
1
st
(p < 0.01), 2
nd
(p<0.05), 3rd (p < 0.05), 5
th
(p < 0.05),
6th (p < 0.01), and 7
th
(p < 0.001) days. The higher dose
showed comparable edema inhibition with the standard
drug at D
2
, D
4
, D
5
, D
6
and D
7
, while its effect was higher
at D
1
and lower at D
3
(Figure 2).
Discussion
Considering the socioeconomic impacts of pain and inam-
mation and having the knowledge potential herbal medicines
from traditionally claimed plants, the need for searching
Table 2 Effect of 80% Hydromethanolic Root Extract of Echinops kebericho on Hot Plate Latency Time in Mice
Treatment Groups 0M 30M 60M 90M 120M
MRP 3.26 ± 0.26 10.0± 0.24b
3
c
3
d
3
10.65± 0.30b
3
c
2
d
1
10.98± 0.14b
3
c
3
d
2
11.10± 0.28b
3
cd
DW 2.95 ± 0.27 3.42 ± 0.11 3.75±0.09 3.52 ± 0.10 3.24± 0.05
100 mg/kg 3.47 ± 0. 101 8.19± 0.23a
3
b
2
9.29± 0.19a
2
b
3
8.83± 0.20a
3
b
3
d
2
e
3
9.58 ± 0.35ab
3
200 mg/kg 3.35 ± 0.11 8.56± 0.45a
3
b
2
9.73± 0.27a
2
b
3
10.02± 0.26ab
3
c
2
10.31 ± 0 0.38b
3
400 mg/kg 2.98 ± 0. 17 9.16 ± 0.50b
3
10.11±.30b
3
10.40± 0.26b
3
c
3
10.59 ± 0.39b
3
Notes: Values are expressed as Mean ± S.E.M (n = 6); analysis was performed with One-Way ANOVA followed by Tukey post hoc test for multiple comparison. a = as
compared to +ve control, b = as compared to -ve control, c = as compared to 100 mg/kg EK, d = as compared to 200 mg/kg EK, e = as compared to 400 mg/kg EK, 3p <
0.001, 2p < 0.01, 1p < 0.05.
Abbreviations: MRP, morphine (10 mg/kg); DW, distilled water (10 mL/kg); 100 mg/kg, Echinops kebericho extract 100 mg/kg; 200 mg/kg, Echinops kebericho extract 200 mg/
kg; 400 mg/kg, Echinops kebericho extract 400 mg/kg.
0
10
20
30
40
50
60
70
80
30M 60M 90M 120M
ytivitcaciseglanaegatnecreP
Times in minute
MRP 10mg/kg
DW 10mg/kg
EK 100mg/kg
EK 200mg/kg
EK 400mg/kg
Figure 1 Percentage analgesic activity of 80% hydromethanolic root extract of Echinops kebericho in thermal-induced latency in mice. Analysis was performed with One-Way
ANOVA followed by Tukey post hoc multiple comparison test. Data was expressed in mean ± SEM. N = 6.
Abbreviations: MPR, morphine (10 mg/kg); DW, distilled water (10 mL/kg); EK 100 mg, Echinops kebericho extract (100 mg/kg); EK200 mg, Echinops kebericho extract
(200 mg/kg); EK400, Echinops kebericho extract (400 mg/kg).
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effective antipain and anti-inammatory drugs with minimal
untoward effects from traditional medicinal plants seems
reasonable.
7,25
Thus, searching for medicinal plants which
have been widely used in the community to treat different
pain conditions and inammation with antipyretic activities
are essential concern in this regard. Echinops kebericho is
among the widely used traditional medicinal plants in
Ethiopian folk medicine for treating pain, and different
inammatory conditions.
14,15
However, no any scientic
reports have been found in the literature about its analgesic
and anti-inammatory activities in experimental animal
models so far. It may therefore be worthwhile scientically
investigating the analgesic and anti-inammatory activities
of the root extract of E. kebericho M. in the mice model to
substantiate its claimed traditional use.
Acetic acid-induced writhing test was selected to detect
the peripheral analgesic activity of the extract. Because of its
sensitivity and ability to detect antinociceptive effects of
natural products and test compounds at dose levels which
remains inactive for other methods, acetic acid-induced wri-
thing test is a well recommended model for screening the
peripheral analgesic potentials of test compounds.
26
Intraperitoneal injection of acetic acid causes irritation and
stimulation of the peritoneal cavity that triggers the synthesis
and release of various endogenous inammatory mediators
such as histamine, serotonin, bradykinin substance P, and
PGs.
27
These various endogenous inammatory mediators
elicited chemical-induced visceral pain which is character-
ized by constriction of abdominal muscles together with the
extension of the forelimbs and elongation of the body. That is
why the acetic acid-induced writhing test is considered as
a model of visceral pain.
28
This model has also been asso-
ciated with increased level of PGE and PGF
2
a. Increasing PG
levels within the peritoneal cavity enhances inammatory
pain by increasing capillary permeability and activating pri-
mary afferent nociceptors.
29
Echinops kebericho extract at all doses employed (100,
200 and 400 mg/kg) signicantly (p < 0.01, and p < 0.001)
showed peripheral analgesic activities by reducing the
number of writhing with the respective values of
31.01%, 48.43%, 57.84% as compared with the negative
control. These ndings conrmed that the peripheral
analgesic activity of the extract increased from the lower
dose (100 mg/kg) to the higher dose (400 mg/kg) in dose
Table 4 Effect of 80% Hydromethanolic Root Extract of Echinops kebericho in Formalin-Induced Paw Edema in Mice
Edema Volume (Mean ± SEM)
Groups Day1 Day2 Day3 Day4 Day5 Day6 Day7
DICL10 mg/kg 0.25 ± 0.02b
3
c
2
0.23 ± 0.01b3c
1
0.21±0.007b
3
c
3
d
3
e
1
0.21 ± 0.008b
3
0.16 ± 0.007b
3
c
2
0.13 ± 0.009b
3
c
2
0 0.1± 0.005b
3
c
3
d
1
DW10 ml/kg 0.41± 0.012 0.41 ± 0.016 0.50 ± 0.009 0.49 ± 0.013 0.50 ± 0.009 0.60 ± 0.008 0.50 ± 0.009
EK100 mg/kg 0.30 ±0.008a
2
b
3
e
1
0.26 ±0.007a
1
b
3
e
1
0.30 ±0.007a
3
b
3
e
2
0.22 ± 0.007b
3
0.20 ± 0.007a
2
b
3
e
1
0.18 ± 0.009a
2
b
3
e
1
0.15 ± 0.008a
3
b
c
e
3
EK200 mg/kg 0.28 ±0.009b
3
0.24 ±0.007b
3
0.28 ± 0.007a
3
b
3
0.23 ± 0.007b
3
0.18 ± 0.008b
3
0.17 ± 0.008b
3
0.13 ± 0.007a
1
b
3
EK400 mg/kg 0.24 ±0.009b
3
c
1
0.21 ±0.008b
3
c
1
0.25 ± 0.02a
1
b
3
c
2
0.20 ± 0.004b
3
0.17 ± 0.008b
3
c
1
0.13 ± 0.008b
3
c
2
0.10 ± 0.004b
3
c
3
Notes: Analysis was performed with One-Way ANOVA followed by Tukey post hoc test for multiple comparisons. Data was expressed as mean ± SEM. N = 6. a = compare
to diclofenac, b = compare to the control, c = compare to 100 mg/kg, d = compare to 200 mg/kg, e = compare to 400 mg/kg. 3p < 0.001, 2p < 0.01, 1p < 0.05.
Abbreviations: DICL, (diclofenac 10 mg/kg); DW, distilled water (10 mL/kg); EK 100 mg, Echinops kebericho extract (100 mg/kg); EK200mg, Echinops kebericho extract
(200 mg/kg); EK400, Echinops kebericho extract (400 mg/kg).
Table 3 Effect of 80% Hydromethanolic Root Extract of Echinops kebericho on Carrageenan-Induced Paw Edema Model in Mice
Change in Edema Volume (mL) Mean ± SEM
Treatment Groups 0hr 1hr 2hr 3hr 4hr
IND25 mg/kg 0.46 ± 0.03 0.22 ± 0.005b
3
(51.48) 0.19 ± 0.006b
3
c
2
(56.06) 0.18 ± 0.006b
3
(69.88) 0.12± 0.006b
3
c
3
d
2
(71.54)
DW10 mg/kg 0.45 ± 0.04 0.46 ± 0.01 0.44 ± 0.008 0.59 ± 0.03 0.43 ± 0.01
EK100 mg/kg 0.40 ± 0.02 0.27 ± 0.02b
3
(41.18) 0.25 ± 0.01a
2
b
3
(43.18) 0.21 ± 0.01b
3
(64.77) 0.20 ± 0.01a
3
b
3
e
3
(54.64)
Ek200 mg/kg 0.40 ± 0.02 0.26 ± 0.02b
3
(41.91) 0.22 ± 0.01b
3
(49.62) 0.20 ± 0.01b
3
(66.48) 0.17 ± 0.004b
3
e
1
(61.54)
EK400 mg/kg 0.42 ± 0.02 0.26 ± 0.02b
3
(42.65) 0.19 ± 0.04b
3
c
2
(56.04) 0.17 ± 0.04b
3
(69.57) 0.13 ± 0.002b
3
c
3
d
2
(70.00)
Notes: Values are expressed as Mean ± S.E.M (n = 6); analysis was performed with One-Way ANOVA followed by Tukey post Hoc multiple comparison test. a = as compare to
+ve control, b = as compare to -ve control, c = as compare to 100 mg/kg EK, d = as compare to 200 mg/kg EK, e = as compare to 400 mg/kg EK, 3p < 0.001, 2p < 0.01, 1p < 0.05.
The data in parenthesis showed the percentage inhibition of paw edema.
Abbreviations: IND, Indomethacin (25 mg/kg); DW, distilled water (10 mL/kg); EK100mg/kg, Echinops kebericho extract 100 mg/kg; EK200 mg/kg, Echinops kebericho extract
200 mg/kg; EK400 mg/kg, Echinops kebericho extract 400 mg/kg.
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dependent manner. The increase in analgesic activity with
increasing doses of the extract might be attributed with an
increase in concentration of phytoconstituents that possess
analgesic activity with the maximum dose.
The possible mechanism by which the extract produced
peripheral analgesia in this model might be associated with
inhibiting the synthesis and release of various endogenous
inammatory mediators and suppression of sensitivity of
peripheral nociceptors in the peritoneal free nerve endings
for chemical-induced pain. These proposed mechanisms
are in line with the principles that stated, any agent that
decreases the number of writhing will demonstrate analge-
sia by inhibiting the synthesis and release of PGs, and by
inhibiting the peripheral pain transmission.
28,30
The second model used was the hot plate test, in which
the supra-spinal nociception and the involvement of cen-
tral antinociceptive mechanism were detected.
28
Since the
paws of mice are very sensitive to heat at temperatures
which are not damaging the skin, the central antinocicep-
tive mechanisms of the extract were detected by introdu-
cing the mice to the constantly heated plate and by
observing the reaction times, namely jumping, withdrawal,
and licking of the paws. The time until these responses
occured was prolonged after administration of centrally-
acting analgesics.
31
This model was selected to evaluate the central analge-
sic potential of the extract because of its sensitivity to
strong analgesics, limited tissue damage with a cutoff
time of 15 sec, which is usually applied to limit the
amount of time the mouse spends on the hot plate. The
model also requires less time and measurements are
usually accurate.
28
The extract at all test doses (100 mg/kg, 200 mg/kg
and 400 mg/kg) signicantly (p < 0.05, p < 0.01, p <
0.001 respectively) elevated the pain threshold by increas-
ing the reaction time starting from 30 min of observation
onwards as compared to the negative control. The max-
imum analgesic effects of the extract were observed at
120 min of observation time with their respective percen-
tage values of 63.93%, 66.94%, 69.40% as compared with
morphine (10 mg/kg) which showed a percentage analge-
sic value of 70.81% at this time. At all times of
0
10
20
30
40
50
60
70
80
90
D1 D2 D3 D4 D5 D6
noitibihniamedeegatnecrep
times in days
DW 10ml/kg
EK100 mg/kg
EK 200mg/kg
EK 400mg/kg
DIC 10mg/kg
Figure 2 Percent Edema inhibition of 80% hydromethanolic root extract of Echinops kebericho in formalin-induced paw edema in mice. Analysis was performed with One-
Way ANOVA followed by Tukey post hoc multiple comparison test. Data was expressed in mean ± SEM. N = 6, DICL= (diclofenac 10 mg/kg).
Abbreviations: DW, distilled water (10 mL/kg); EK 100mg, Echinops kebericho extract (100 mg/kg); EK200 mg, Echinops kebericho extract (200 mg/kg); EK400, Echinops
kebericho extract (400 mg/kg).
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observation, the doses of the extract showed analgesic
activities in dose dependent manner. The doses of the
extract took longer time (peak time) to attain for maximal
effect, which for all doses was 120 min. This delay may
be due to a probable time lag for the drug to enter in to the
central compartment and distribute into the target site or
formation of active metabolites that are endowed with
analgesic activity with better half-life. A relatively better
action of 400 mg/kg at all observation time may be
attributed by the presence of high concentration of active
metabolites.
The possibly proposed mechanism of central analgesic
effects of the extract may be by activating the periaque-
ductal gray matter (PAG) to release endogenous peptides
(i.e., endorphin or enkephalin). These endogenous peptides
descend the spinal cord and function as inhibitors of the
pain impulse transmission at the synapse in the dorsal horn
or via peripheral mechanisms involved in the inhibition of
PG, leukotriene, and other endogenous substances that are
key players in central pain transmission.
32
Previous phytochemical screening on EK has revealed
the presence of alkaloids, polyphenols, saponins, phytos-
terols, carotenoids, lignans, sesquiterpene alcohols, acety-
lenic and thiophene compounds, terpenoids, and essential
oil.
33
It is proved that the root extract of EK contained
secondary metabolites including saponin, tannin, alkaloids,
phenols, avonoids, glycosides and steroids by prelimin-
ary phytochemical screening in the present study. So, it
can be said that the analgesic effects shown by the extract
may be due to the presence of these aforementioned and
currently identied phytoconstituents.
34
This suggestion is
in line with the reports that stated, phytoconstituents like,
alkaloids, avonoids, steroids, and tannin isolated from
medicinal plants have been reported to possess
a signicant analgesic activity.
9,28
In the study of the anti-inammatory activity, the root
extract of EK was detected against carrageenan-induced
acute and formalin-induced sub-acute phases of inamma-
tion. Since the inammatory response is a polyphasic tis-
sue reaction, which involves both short lived increase in
vascular permeability and prolonged cellular inltration
and proliferation, it is important to evaluate the potential
of the extract for anti-inammatory effect via a sequential
test valid for various phases of inammation.
9
Carrageenan-induced hind paw edema is a prototype
model which is employed to evaluate the anti-inammatory
potentials of various natural and synthetic products as well as
to determine the possible mechanisms involved in
inammation.
4,14
Carrageenan is a phlogistic, non-antigenic
agent and is devoid of apparent systemic effect, it is also
believed that the experimental model exhibited a high degree
of reproducibility in acute phase inammation. Thus, carra-
geenan-induced paw edema, is a frequently used method for
the screening of acute inammatory potentials of various
natural products.
14
In the present study, acute inammation was induced by
sub planar injection of carrageenan (1%v/v in normal saline)
in the left hind paws of the mice. Following the induction of
carrageenan, an acute localized inammation was induced
through sequential release of various endogenous inamma-
tory mediators. The release of these endogenous mediators
was biphasic. The early phase (0 and 2.5 h) after carrageenan
induction is mainly mediated by the release of histamine,
serotonin, and bradykinin. These mediators are attributed for
inammation by increasing vascular permeability in the
damaged tissue surroundings. The late phase which is
sustained by the overproduction of COX-2 and its pro-
inammatory PGs product, with inltration of polymorpho-
nuclear leucocytes (neutrophils), took place 2.5–6hrs post
carrageenan induction.
9
There are also other chemicals med-
iators released during the late phase of inammation such as,
oxygen-derived free radicals like superoxide anion (O
2-
) and
hydroxyl radicals (OH
−
), nitric oxide (NO) which play an
important role in the development and progression of acute
inammation.
12,35
The extract at all test doses employed (100, 200 and
400 mg/kg) signicantly (p < 0.05, p < 0.01 and p < 0.001
respectively) decreased the formation of edema starting
from 1 hr post carrageenan induction and the effects per-
sisted (p < 0.001) till the 4
th
hr of observation. The effect
of the extract started from the 1
st
phase (1 hr) and con-
tinued till the 4
th
hr (second phase) of inammation. This
observation suggested that bioactive constituents in the
extract may suppress both phases of acute inammation
by interfering with the release and/or activity of the che-
mical mediators.
The maximum percentage of edema inhibition by all
doses of the extract was observed at the 4
th
time of observa-
tion with the respective values of 54.65%, 61.54% and
70.00%. These effects veried that the extract’s anti-
inammatory effect was in dose dependent manner. Edema
inhibition potential showed by the higher dose of the extract
(400 mg/kg) was comparable with that of the standard drug
(Indomethacin 25mg/kg) with the respective values of
70.00% and 71.54% at the 4
th
time of observation.
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The signicant anti-inammatory effects shown by
both the extract and the standard drug (Indomethacin
25 mg/kg) evidenced that their effects started in the early
phase of inammation by inhibiting endogenous inam-
matory mediators such as serotonin and histamine as they
involve in the early phase of inammation, while the
effects of edema inhibition reached maximum at the 4
th
time suggests that both the extract and the standard drug
have profound anti-inammatory effects against various
endogenous inammatory mediators that involve in the
late phase of inammation such as, COX, different PG
analogues, BK and/or leukotriene or they could have, free
radical scavenging activities.
8,36
Formaldehyde-induced paw edema model is the well-
known model for the evaluation of anti-arthritic and anti-
inammatory potentials of natural products in sub-acute
phase of inammation. Injection of 2% formalin v/v with
distilled water subcutaneously in the palm of the mice in the
1
st
and 3
rd
days of observation produced progressive paw
edema which was characterized by increased migration of
leucocytes and phagocytes, inltrations of neutrophils,
macrophages and proliferation of broblasts into the sur-
rounding of injured area.
35
Hence, the inhibition of formal-
dehyde-induced edema is one of the most prototype methods
to evaluate anti-inammatory activity in sub-acute phase of
inammation, anti-proliferative activity, and screening anti-
arthritic agents from natural products
35,36
In this model the three doses of the extract (100, 200
and 400 mg/kg) showed signicantly mice paw swelling
inhibition (p < 0.05, p < 0.01, p < 0.001 respectively)
starting from the 1
st
day of observation and the effects
progressed (p < 0.01 and p < 0.001) till the 7
th
day. The
effect shown by the extract was in dose dependent manner.
The highest percentage of edema inhibition for all doses
(100, 200 and 400 mg) was observed at the 7
th
day of
observation with the respective values of 69.46%, 74.83%
and 79.87% respectively. The highest dose of the extract
showed signicantly higher effect (p < 0.05 and p < 0.01) via
the lower (100 mg/kg) and the middle dose (200 mg/kg)
while its effect was higher at the 1
st
and 2
nd
days and remain
comparable at the rest days of observation with the standard
drug (diclofenac 10 mg/kg). Increasing edema inhibition of
the extract as the dose increased can be explained by the
possible existence of an adequate concentration of the active
metabolite(s) in the maximum dose level (400 mg/kg) when
compared to the lower and middle (100 and 200 mg/kg) dose
levels of the extract and by the quick metabolism and elim-
ination of the effective phytoconstituents present in
inadequate concentrations in the lower and middle dose
levels. The observed effects of the extract may be through
anti-proliferative activities against broblasts, inhibition of
inltrations of neutrophils and macrophages, and antagoniz-
ing the migration of leucocytes and phagocytes in to the area
of inammation.
35
The anti-inammatory action of E. kebericho extract
in the present study can be supported by previous reports
from scientic journals that stated plants which contain
mainly alkaloids, avonoids, saponin, and tannins phe-
nolic compound, glycosides, coumarins and triterpenoid
chemical constituents showed strong anti-inammatory
effects. So, it can be deduced that the anti-inammatory
effect of E. kebericho extract in the present study may be
due to the presence of alkaloids, avonoids, saponin,
tannin, and triterpenoids. Alkaloids exert its anti-
inammatory activity through interfering with indubita-
ble COX expression and production of PGE
2
, inhibition
of pro-inammatory cytokines production like IL-1β, IL-
6, TNF-α, terpenoids exert its anti-inammatory effect
through inhibition of PLA
2
activity, inhibition of TNF-α
production, inhibition of iNOS expression, inhibition of
COX-2 expression, and inhibition of NF-κB activation,
while saponins are believed to interfere with iNOS
expression, inhibition of COX-2 expression and subse-
quent production of PGE
2
, and exert their sequential
anti-inammatory effects.
4,26
Furthermore, polyphenols
exert their anti-inammatory properties through inhibi-
tion of the production of inammatory cytokines and
chemokine and suppressing the activity of (COX and
iNOS and thereby decreasing the production of reactive
oxygen and nitrogen species).
8
The results obtained from the present study were in
line with the ndings of others
8,9,28
that demonstrated the
analgesic and anti-inammatory activities of medicinal
plants in a dose dependent manner.
In general, it can be concluded that the anti-
inammatory activity of E. kebericho extract may be due
to cumulative effects of the presence of different active
phytoconstituents in reducing the synthesis, release and
action of different endogenous inammatory mediators
that are mentioned above which play key roles for the
development and progress of both acute and sub-acute
inammation.
Conclusions
In conclusion, the plant extract possessed peripheral
analgesic activity and central pain inhibition potential. It
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also exhibited an anti-inammatory activity, in both acute
and sub-acute phases of inammation. These results might
imply that the plant extract was involved in inhibition of
various endogenous inammatory mediators, pain trans-
mission and mediators, due to the presence of secondary
metabolites including alkaloids, avonoids, saponins, ter-
penoids, tannins, and essential oils which are repeatedly
reported to possess analgesic and anti-inammatory activ-
ities. The current ndings put scientic evidence about the
traditional claimed uses of Echinops kebericho M. for
painful conditions and inammation purpose in Ethiopian
folk medicines.
Further investigations should be conducted on the frac-
tionation to determine the most active fraction, constituent
isolation, binding studies and electrophysiological proce-
dures may also be useful to fully elucidate anti pain and
anti-inammatory and specic mechanisms of
E. Kebericho related to these mechanisms.
Abbreviations
ASA, acetyl salicylic acid; EK, Echinops kebericho M;
CNS, central nervous system; COX, cyclooxygenase; IL,
interleukin; iNOS, inducible nitric oxide synthase;
NSAID, nonsteroidal anti-inammatory drug; NF-κ,
nuclear factor kappa; PGs, prostaglandins; TNF, tumor
necrosis factor.
Data Sharing Statement
The datasets used and or analyzed during the current work
are available from the corresponding author up on a rea-
sonable request.
Ethics Approval and Consent
Ethical clearance was obtained from Department of
Pharmacology, School of Pharmacy Collage of Medicine
and Health Science University of Gondar.
Acknowledgment
We would like to acknowledge Debre-tabor University for
providing nancial support. We are also grateful to Mr
Zewudu Birhanu (B. pharm, MSc, associate professor) for
providing carrageenan.
Author Contributions
All authors contributed to data analysis, drafting or revis-
ing the article, have agreed on the journal to which the
article will be submitted, gave nal approval of the version
to be published, and agree to be accountable for all aspects
of the work. TY conceived the idea, drapted the proposal,
collected the plant matterials and conducted the actual
laboratory work. TY and YKE prepared and critically
reviewed the nal manuscript for publication. EMB,
YKE, MG and MA were involved in the design and
implementation stage of the study, and revising the the
manuscript critically for important intellectual content. All
authors read and approved the nal version of the
manuscript.
Disclosure
The authors report no conicts of interest in this work.
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