Balkan Med J 2020;37:189-95 Original Article 189
Antinociceptive Action of Moringa peregrina is Mediated by an
Interaction with α2-Adrenergic Receptor
1Department of Biological Sciences, Jordan University School of Science, Amman, Jordan
2Department of Pharmaceutical Sciences, Al-Ahliyya Amman University School of Pharmacy, Amman, Jordan
3Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan
Sahar M. Jaffal1, Belal O. Al-Najjar2, Manal A. Abbas3, Sawsan A. Oran1
Background: Moringa peregrina (M. peregrina) is an edible,
drought-resistant tree that is native to semi-arid countries. It is used as
a painkiller in folk medicine.
Aims: To study the antinociceptive effects of the leaf extract of
M. peregrina in mice.
Study Design: Animal experimentation.
Methods: We employed thermal (hot plate and tail-immersion tests)
and chemical (writhing and formalin tests) pain models in male
BALB/c mice (eight animals per group) to investigate the mechanisms
involved in the antinociceptive actions of M. peregrina. Additionally,
we identified the chemical constituents present in the extract of
M. peregrina by using liquid chromatography-mass spectrometry
analysis, and predicted the possible active constituents that interact
with the receptor based on molecular docking simulations.
Results: In the writhing test, 200 mg/kg of M. peregrina extract
restricted abdominal cramps by up to 55.97% (p<0.001). Further,
it reduced the time of paw-licking in the early and late phases of
formalin test by up to 56.8% and 65.5%, respectively, as compared to
the percentage inhibitions of 50.5% and 48.4% produced by 30 mg/kg
diclofenac sodium in the early and late phases, respectively (p<0.05).
This effect was abrogated by yohimbine (1 mg/kg, intraperitoneally),
but not by methysergide (5 mg/kg, intraperitoneally), in the late phase
only, which indicates that the action of M. peregrina in formalin
test is not mediated by 5-HT2 serotonin receptors, but rather via α2-
adrenergic receptors. In the hot plate test, but not on tail-immersion
test, the high dose (400 mg/kg) of the extract increased the latency
time after 30 minutes of its administration. Yohimbine antagonized
the action of M. peregrina in the hot plate test. Based on LC-MS
analysis, the major constituents found in M. peregrina methanolic
extract were chrysoeriol 7-O-diglucoside, lupeol acetate, quercetin,
and rutin. Depending on the molecular docking results, the activity
of M. peregrina extract could be due to the binding of chrysoeriol
7-O-diglucoside, quercetin, and rutin to the α2-adrenergic receptor.
Conclusion: Interaction with the α2-adrenergic receptor serves as a
possible mechanism of the M. peregrina analgesic effect.
Keywords: Adrenergic alpha-2 receptor antagonists, analgesics,
molecular docking, Moringa peregrina
Moringa (Moringaceae) is a genus that comprises 13 species of trees
and shrubs growing in the tropical and sub-tropical regions of our
world (1). Various civilizations, such as Greek, Indian, and Egyptian,
have utilized Moringa for many millennia. Moringa (mainly its
leaves and fruits) has always been an integral part of people’s diet.
Additionally, Moringa leaves were fed to the ancient Mauryan
warriors of India to provide them with energy and relieve their pain
(2). Moringa peregrina (M. peregrina) is a drought-resistant tree that
grows in the dry or semi-arid countries located near the Red Sea such
as Somalia, Syria, Palestine, and Yemen (3). The tree is known for
its fast growth and can reach up to 3-10 meters in height after ten
months of its cultivation (4). The leaves of M. peregrina are obovate,
alternate, deciduous, and about 30-40 cm in length (2). Almost all
parts of this plant are consumed by humans as a vegetable due to its
taste, flavor, and nutritional value. The leaves of M. peregrina are
great sources of proteins, vital elements (Ca
essential amino acids, and vitamins such as vitamin A, C, and E (5,6).
M. peregrina is considerably used by people of various cultures
in traditional healing practices during childbirth and for treating
multiple disorders, such as malaria, fever, diabetes, abdominal pain,
asthma, headache, constipation, muscle pain, hypertension, burns
(7,8). Pharmacological studies have reported that M. peregrina
has antimicrobial (9), in vitro antioxidant (8), anticancer (10), and
antispasmodic properties (7) as well as immunomodulatory activities
(both ex vivo and in vivo) (3). In rats, M. peregrina has demonstrated
anti-inflammatory (11), antiulcer (12), antihyperglycemic (13),
antihyperlipidemic (14), antihypertensive effects (15), and memory-
enhancing activities (16).
Address for Correspondence: Manal A. Abbas, Department of Biological Sciences, Jordan University School of Science, Amman, Jordan
Phone: +962 0777 405887 e-mail: firstname.lastname@example.org ORCID: orcid.org/0000-0002-8962-1879
Received: 04 November 2019 Accepted: 02 March 2020 • DOI: 10.4274/balkanmedj.galenos.2020.2019.11.14
Available at www.balkanmedicaljournal.org
Cite this article as:
Jaffal SM, Al-Najjar BO, Abbas MA, Oran SA. Antinociceptive Action of Moringa peregrina is Mediated by an Interaction with α2-Adrenergic Receptor.
Balkan Med J 2020;37:189-95
©Copyright 2020 by Trakya University Faculty of Medicine / The Balkan Medical Journal published by Galenos Publishing House.
Despite the use of M. peregrina in folk medicine as a painkiller
(4), there is a paucity of detailed studies determining the analgesic
effects of this plant. Previous work on M. peregrina includes only
a preliminary investigation that employed writhing and hot plate
tests (12). The purpose of this investigative study is to examine
the effects of M. peregrina on the early and late phases of formalin
test. Additionally, we performed tail-immersion test to study
the effects of M. peregrina at the spinal level. Furthermore, we
studied the mechanism by which M. peregrina exerts its action.
MATERIALS AND METHODS
Diclofenac sodium was obtained from Novartis, Switzerland,
whereas methysergide was obtained from Sigma-Aldrich, USA.
Yohimbine was purchased from Tocris Bioscience (UK). All the
drugs were freshly prepared in a sterile normal saline solution and
administered intraperitoneally (i.p).
Collection of plant material
The leaves of M. peregrina were collected in June 2012 from Wadi
Bin-Hammad Valley (Karak, south of Jordan). Professor Sawsan
Oran, plant taxonomist at the University of Jordan, authenticated
Preparation of plant extract
The dried leaves of M. peregrina were extracted by maceration
in 96% methanol (Scharlau Chemie, Spain). Rotary evaporator
was used to evaporate methanol under reduced pressure and at a
temperature not more than 45oC. The extract was stored at -20°C,
and it was freshly prepared, before its use, by dissolving it in the
sterile normal saline solution.
All employed procedures in this study complied with the
guidelines of the International Association for the Study of Pain
(17) and were approved by the ethical committee at Al-Ahliyya
Amman University (ethical approval no. AAU-2/4/2018). Male
BALB/c mice (weight: 20-25 g) were kept at standard laboratory
conditions (at 23±2
C in both dark and light environments
consecutively). Food and water were provided ad libitum.
The animals were allowed to acclimatize to the experimental
laboratory conditions for 120 minutes before the commencement
Pretreatment of animals
In all the experiments, animals were pretreated i.p with 10 mL/
kg vehicle (sterile normal saline solution, control) along with 200
mg/kg or 400 mg/kg M. peregrina methanolic extract 30 minutes
before the commencement of the experiments. Diclofenac sodium
(30 mg/kg) was used as a standard drug similar to the research
of Jaffal and Abbas (18). Methysergide (5 mg/kg) or yohimbine
(1 mg/kg) was injected 15 minutes before the administration of
a fixed dose of M. peregrina extract (200 mg/kg or 400 mg/kg).
The choice of doses of antagonists was based on the previously
published studies (19,20).
Writhing test was conducted, as per Koster method (21), via
the administration of acetic acid solution (1%, 10 mL/kg) i.p in
the animals 30 minutes after receiving the vehicle solution, M.
peregrina extract (200 mg/kg or 400 mg/kg), or diclofenac sodium.
Each group consisted of eight animals (mice). After 10 minutes
of acetic acid administration, the number of writhes was counted
for 20 minutes. A writhe is considered as a contraction of the
abdominal muscles accompanied by the elongation of the body and
extension of the forelimbs. The percentage inhibition of abdominal
cramps was calculated by using the following formula:
% inhibition = Average number of writhes in control - Average
number of writhes in treated animals × 100%
Average number of writhes in control.
Paw-licking test was performed after the intraplantar administration
of 2.5% formalin (20 µL) to the left hind paw of the mouse. The total
time spent in licking the injected paw, lifting the leg, or exhibiting
a flinching behavior were recorded in the first 5 minutes after the
administration of formalin (early phase) and during the late phase
(25-30 minutes after injection). The percentage inhibition was
calculated according to the following formula:
% inhibition= Average time of licking in control - average time of
licking in treated animals × 100%
Average time of licking in control
Hot plate test
The hot plate test was used to determine latencies in pain reaction.
Mice reactions were assessed by individually placing the animals
into a transparent container on a hot plate at 55±1oC. Each mouse
underwent this procedure only once. The time between the animal’s
placement and first jump was recorded as a measure for the latency
of pain reaction. A cut-off time of 60 seconds was determined to
avoid tissue damage.
Tail-immersion test was performed by dipping the tail in water at
55±1oC. The time starting from immersing the tail in water till the
appearance of the first flick was calculated. A cut-off time of 10
seconds was determined.
Liquid chromatography-mass spectrometry (LC-MS)
LC-MS separation was performed in the mobile phase. This phase
have solvents A and B in gradient, in which A contained 0.1%
(v/v) formic acid in water and B had 0.1% (v/v) formic acid in
acetonitrile. Agilent ZORBAX Eclipse XDB-C18 column (2.1×150
mm ×3.5 μm) was used in this procedure. The oven was set at 25
̊C, and the volume of injection was 1 μL containing 18 mg/mL in
methanol. We used Shimadzu LC-MS 8030 with electrospray ion
mass spectrometer (ESI-MS) to monitor the eluent under positive
ion mode. Then, we scanned it from 100 to 1,000 mass/number of
ions (m/z). ESI was performed by using skimmer 65 V and at a
fragmentor voltage of 125 V. Highly pure (99.99%) nitrogen was
used as drying gas at 10 L/min flow rate, capillary temperature
at 350 ̊C, and nebulizer at 45 psi. The sample was injected to the
Balkan Med J, Vol. 37, No.4, 2020
Jaffal et al. Antinociceptive Action of Moringa peregrina
mass detector by using the LC-30AD pump, Shimadzu CBM-
20A system controller, cooler, and CTO-30 column oven with the
SIL-30AC autosampler. The results were validated by running the
authentic standard compounds and referring to the literature as in
the research of (16).
Protein preparation and homology modeling
The homology model for α2-adrenergic receptor was developed
from the SWISS-MODEL server (22). Other software included in
this study were Discovery Studio Visualizer 4.0 (Accelrys Software
Inc, San Diego; http://www.accelrys.com), ACD/ChemSketch,
(www.acdlabs.com), and AutoDock4 (23).
The protein sequence of α2-adrenergic receptor was selected from
the universal protein source under the code no. P08913. The
homology model of the protein was built at the SWISS-MODEL
web server program. Initially, BLAST (24) was used to search for
the target sequences against the primary amino acid sequence.
Thereafter, the quality of the template was predicted from the
target-template alignment features. Further, the best-quality
templates were chosen for building the model.
ACD/ChemSketch software was used to construct the chemical
structures of major compounds in M. peregrina extract. These
compounds were chrysoeriol 7-O-diglucoside, lupeol acetate,
quercetin, and rutin. The compounds were drawn and saved as MOL
files by ChemSketch software and then converted to PDB files.
Ligand files in the PDB format were prepared by AutoDockTools.
Each compound was opened separately, charges were added, and all
the hydrogen atoms were merged. Molecular docking simulations
of the compounds were performed by utilizing AutoDock 4.2.
Kollman and Gasteiger charges were added to both proteins and
plant compounds, respectively. A set of grid maps were created by
using AutoGrid 4 (The Scripps Research Institute, San Diego, CA,
USA). Then, a grid box was utilized to select the area of the protein
structure to be mapped. The box size was set to 22.5, 22.5, and 22.5
Å (x, y, and z, respectively). Energy optimization and minimization
was conducted by applying Lamarckian genetic algorithm in the
docking simulation (16).
All data of this study passed the normality test (Shapiro-Wilk test).
Brown-Forsythe and Welch One-Way analysis of variance tests for
parametric analysis were used to examine the statistical difference
between the groups. Version 6 of GraphPad Prism was chosen to
perform the statistical analysis of this study.
In the writhing test, 200 mg/kg and 400 mg/kg of M. peregrina
extract inhibited abdominal cramps by up to 55.97% and
88.00%, respectively, as compared to inhibition percentage of
47.69% produced by 30 mg/kg of diclofenac sodium. Yohimbine
antagonized the action of M. peregrina in the writhing test
(Table 1). Additionally, the methanolic extract of M. peregrina
reduced the time of paw-licking in the early and late phases of
formalin test. high (400 mg/kg) and low (200 mg/kg) doses of
M. peregrina extract exhibited inhibition percentages of 59.6%
and 56.8%, respectively, whereas 30 mg/kg diclofenac sodium
produced an inhibition percentage of only 50.5% in the early
phase of formalin test (Figure 1A). High (400 mg/kg) and low
(200 mg/kg) doses of M. peregrina extract exhibited inhibition
percentages of 50.1% and 65.5%, respectively, as compared to an
inhibition percentage of 48.4% achieved by 30 mg/kg diclofenac
sodium in the late phase of formalin test (Figure 1B). This effect
(in late phase only) was abrogated by yohimbine, but not by
In the hot plate, but not tail-immersion test, a high dose of M.
peregrina (400 mg/kg) extract increased the latency time after 30
minutes of its administration. Yohimbine antagonized the action
of M. peregrina in the hot plate test (Table 2). By using LC-MS,
18 compounds were detected in the extract (Table 3). Chrysoeriol
7-O-diglucoside, lupeol acetate, quercetin, and rutin were the
major compounds present in this extract.
SWISS-MODEL had generated around 531 templates, of which the
top 50 filtered templates can be found in the supplementary material
(Table S1). The homology model of α
-adrenergic receptor having
a resolution of 1.96 Å was validated by using the Ramachandran
plot (Figure S1 in supplementary materials). Quality estimate of the
homology model is presented in Figure S2 while predicted local
similarity to target for the homology model is shown in Figure S3 in
supplementary material. Figure 2 shows all the successfully docked
plant constituents against the α
-adrenergic receptor model, and
Table 4 shows the results of lowest binding energies .
The docking results showed a higher binding affinity for the
compounds, namely chrysoeriol 7-O-diglucoside, quercetin,
and rutin, with a low affinity for lupeol acetate. Further
investigation of these compounds in the binding site have
revealed that chrysoeriol 7-O-diglucoside, quercetin, and
rutin participate in the hydrogen bond interactions as well as
hydrophobic interactions within the binding site, whereas
lupeol acetate participates only in the hydrophobic interactions
Balkan Med J, Vol. 37, No.4, 2020
Jaffal et al. Antinociceptive Action of Moringa peregrina 191
TABLE 1. Writhing test results
Yohimbine (1 mg/
kg) and M. peregrina
Yohimbine (1 mg/kg)
and M. peregrina
No of writhing 49.00±3.16 21.57±6.65* 54.80±7.79#5.88±4.22* 18.25±8.88£25.63±7.78*
% inhibition - 55.97% - 88.00% - 47.69%
P value - p<0.0001 p<0.0001 p<0.0001 p<0.05 p<0.0001
n=8 values are mean ± standard deviation of the mean, *Significantly different from control, #Significantly different from M. peregrina (200 mg/kg), £Significantly different from M.
peregrina (400 mg/kg)
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Jaffal et al. Antinociceptive Action of Moringa peregrina
TABLE 2. Results of thermal antinociceptive tests: hot plate and tail flick tests
Yohimbine (1 mg/kg) and
Latency time in hot plate test (sec.) 9.23±1.63 9.46±1.85 30.87±2.21*
Latency time in tail flick test (sec.) 2.92±0.53 2.70±0.60 2.34±0.60 - 4.49*±0.63
n=14 Values are mean ± standard deviation of the mean, *Significantly different from control, #Significantly different from M. peregrina (400 mg/kg)
FIG. 1. a, b. Results of paw-licking test (formalin test). (a) Early phase
(0-5 minutes after injection) (b) Late phase (25-30 minutes after
*Signicant difference from the control (p<0.05), **P<0.05 signicantly
different from M. peregrina (200 mg/kg)
FIG. 2. Solid ribbon representation of α2-adrenergic receptor docked
with the major compounds in M. peregrina extract, namely chrysoeriol
7-O-diglucoside, lupeol acetate, quercetin, and rutin, in the active site
of the receptor.
TABLE 3. Chemical constituents of M. peregrina as detected by liquid
1 Apigenin 0.88
2 Amyrin 10.1
3 Beta-sitosterol-3-d-glucoside 7.1
4 2-Butyl isothiocyanate 1.8
5 Chrysoeriol 7-O-diglucoside 16.3
6 5,5-Dimethyl-3-phenyl-1,3-oxazolidine-2-thione 3.3
7 Lupeol acetate 15.7
8 Methyl Glucosinolate 1.8
9 2-Methylpropyl isothiocyanate 3.1
10 Neochlorogenic acid 0.63
11 Niazirin 0.68
12 Niazirinin 1.8
13 2-Propyl isothiocyanate 2.2
14 Quercetin 13.3
15 Rhamnetin 1.05
16 4(a-L Rhamnosyloxy) benzyl isothiocyanate 0.97
17 Rutin 11.4
18 Sitosterol 6.3
In our study, M. peregrina exhibited marked analgesic properties
induced by acetic acid in the writhing test. The percentage inhibition
produced by 200 mg/kg and 400 mg/kg doses were 55.97% and
88%, resprctively. Similar findings were obtained by Elbatran et
al. (12) in which 113.4 mg/kg of M. peregrina decreased acetic
acid-induced abdominal cramps by 70.7% in mice. Additionally, at
the doses of 100 and 200 mg/kg, M. oleifera extract significantly
decreased writhes (25). This effect was not reversed by naloxone,
thereby indicating a peripheral non-opioid mechanism of action
(26). Bhattacharya et al. (27) also found that M. oleifera extract
exhibited analgesic activity at 100, 200, and 400 mg/kg in acetic
acid-induced abdominal cramps in the writhing test, further
showing percentage inhibitions of 32.21%, 59.71%, and 78.61%
of writhes, respectively, as compared with the control group (27).
M. peregrina leaf extract demonstrated noticeable antinociception
in the early and late phases of the formalin test. The administration
of formalin causes three distinct periods with high licking
behavior: an early phase lasting the first 5 minutes, and a late
phase in the last 5 minutes after the injection. The early phase
(neurogenic pain) is due to the direct activation of the primary
afferent fibers. The late phase is known as inflammatory pain,
thereby representing the effect in the primary afferents and central
sensitization of spinal cord circuits secondary to the events that
occurred during Phase I (28). The lowest dose of M. peregrina
(200 mg/kg) was more active in the late phase than in the early
phase and was more active than the higher dose (400 mg/kg). This
can be explained by the non-specific effect of compounds in a
higher dose with receptors that can block the interaction of active
compounds with the receptor involved in the antinociceptive
action. As per our knowledge, this study is the first one to report
on the activity of M. peregrina in the formalin test. Similarly, M.
oleifera leaves exhibited antinociceptive activity in the late phase
(25,26). Both polar and non-polar extracts of M. oleifera leaves
were effective in both phases of formalin test. Due to the presence
of central and peripheral actions of non-polar active compounds,
the hexane extract was more effective than the ethanolic extract
In the hot plate test, a high dose of M. peregrina (400 mg/kg) extract
increased the latency time after 30 minutes of its administration.
Similar results were reported by Elbatran et al. (12). In search
for the mechanism of antinociceptive action of M. peregrine, the
antagonists for adrenergic and 5-hydroxytryptamine type 2 (5-
HT2) serotonergic receptors were used in this study since previous
studies suggested that the closely related species of M. oleifera
interact with both dopaminergic and 5-HT2 serotonergic receptors
(30). This study showed that yohimbine antagonized the action of
Balkan Med J, Vol. 37, No.4, 2020
Jaffal et al. Antinociceptive Action of Moringa peregrina 193
FIG. 3. a-d. Stick representation of a. chrysoeriol 7-O-diglucoside, b.
lupeol acetate, c. rutin, and d. quercetin in the active site that forms
hydrophobic (pink dots) interactions and hydrogen bond (green dots)
with α2-adrenergic receptor.
TABLE 4. The lowest binding energies obtained from AutoDock 4.2 for plant constituents against α2-adrenergic receptor and the interacting amino acids.
Compound Lowest binding energy (kcal/mol) Interacting amino acids
Chrysoeriol 7-O-diglucoside -8.74 Asn93, Glu94, Arg187, Cys188, Asn191, Ser200, Tyr394, Arg405.
Lupeol acetate -4.01 Leu110, Val186, Cys188, Lys409.
Quercetin -7.88 Ser90, Asn93, Glu94, Asp113, Val186.
Rutin -8.67 Glu94, Asp113, Ile190, Asn191, Tyr394, Trp413, Tyr416.
M. peregrina in the hot plate test. As per our knowledge, this is the
first report that shows the involvement of α2-adrenergic receptors
in the action of M. peregrina. The related species of M. oleifera
showed significant analgesic dose-dependent activity in the hot
plate test (27). Study suggested that the effect of M. oleifera in the
hot plate test is modulated at the central antinociceptive level via
opioid receptors since the use of naloxone (5 mg/kg) reversed the
effect of extract (26).
In the tail-immersion test, M. peregrina extract had no effect on the
latency time of tail immersion after the administration of extract. In
contrast, M. oleifera produced considerable antinociceptive action
by enhancing tail-immersion latency period at 30 minutes (31). This
could be explained by the presence of different phytoconstituents
in the two species.
Both tail-immersion and hot plate tests are thermal acute pain tests.
However, tail-immersion test works at the spinal level, whereas the
hot plate test is a supraspinally controlled test (32). To the best of
our knowledge, this study is the first one to report the lack of spinal
reflexive action of M. peregrina.
Elbatran et al. (12) isolated four flavonoids from the aerial parts
of M. peregrina and found that the major flavonoids are quercetin-
3-0-rutinoside (rutin), quercetin, chrysoeriol-7-0-rhamnoside, and
6,8,3,5-tetramethoxy apigenin. These flavonoids showed both
analgesic and anti-inflammatory properties. Additionally, the
aerial sections of M. peregrina have β-sitosterol-3-O-glucoside,
β-sitosterol, α-amyrin, β-amyrin, lupeol acetate, apigenin,
6-methoxy-acacetin-8-C-β-glucoside, neochlorogenic acid,
rhamnetin, and rhamnetin-3-O-rutinoside (13). Lupeol acetate
isolated from M. peregrine has a well-documented analgesic
Depending on the previous results, we propose that the plant extract
will interact with α
-adrenergic receptor to exert its analgesic activity.
Thus, it will be useful to determine the active compounds that are
responsible for this action. Due to the absence of complete α
receptor crystal structure, homology modeling is considered as an
attractive technique for obtaining the structure. Such technique has
proven to be an appropriate choice to obtain the useful 3D structure
of the protein (34). Furthermore, molecular docking simulations have
shown a good affinity of chrysoeriol 7-O-diglucoside, quercetin, and
rutin toward the active site of the receptor.
In conclusion, our results suggest an interaction with α2-adrenergic
receptor as a possible mechanism of analgesic action of M.
peregrina. Depending on the molecular docking results, the activity
of M. peregrina extract with α2-adrenergic receptor could be due
to the binding of chrysoeriol 7-O-diglucoside, quercetin, and rutin
compounds to the receptor.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: This work was published with the support of Al-Ahliyya
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