Journal of Ethnopharmacology 259 (2020) 112934

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Anti-nociceptive and anti-inflammatory activities of ethanol extract and T

fractions of Morus mesozygia Stapf (Moraceae) leaves and its underlying
mechanisms in rodents
Oluwakemi O. Ariyoa,b, Abayomi M. Ajayia,∗, Benneth Ben-Azua,c, Adegbuyi O. Aderibigbea
a
Neuropharmacology Unit, Department of Pharmacology & Therapeutics, University of Ibadan, Oyo-State, Nigeria
b
Pharmacy Department, Health Services Directorate, Federal University of Agriculture, Abeokuta, Nigeria
c
Department of Pharmacology, Faculty of Basic Medical Sciences, PAMO University of Medical Sciences, Port Harcourt, Rivers State, Nigeria

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacological relevance: Morus mesozygia Stapf (Moraceae), commonly known as African mulberry, is
Morus mesozygia traditionally used for the treatment of inflammatory disorders such as rheumatism and dermatitis.
Moraceae Aim: This work aimed to evaluate the anti-nociceptive and anti-inflammatory effects of its ethanol (EEMm)
Ethylacetate fraction extract, and ethylacetate fraction (EAFMm).
Anti-nociceptive
Methods: The anti-nociceptive and anti-inflammatory effect of ethanol extracts of M. mesozygia (EEMm), and its
Anti-Inflammatory
COX-2
ethylacetate (EAFMm) and residual aqueous fraction (RAFMm) was evaluated in hotplate, acetic acid and for-
NFκB malin tests and as well in membrane stabilizing assay and carrageenan-induced paw oedema models. Mechanism
of anti-inflammation of EAFMm was investigated in the carrageenan-induced air-pouch model.
Results: In the hot plate test of nociception, only the EAFMm showed significant (p < 0.05) anti-nociceptive
activity. The extract and fractions significantly reduced number of writhing with EAFMm (400 mg/kg) showing
highest inhibition (66.5%) in the acetic acid-induced writhing in mice. EEMm and EAFMm (400 mg/kg) sig-
nificantly reduced the paw licking time in the early and late phases of the formalin test. The extract and fractions
showed good membrane stabilizing activity comparable to indomethacin. EAFMm (100 and 400 mg/kg) showed
the highest inhibition of paw oedema (53.4% and 58.1%) in the carrageenan-induced paw oedema model. The
EAFMm (100 and 400 mg/kg) reduced exudate volume relative to carrageenan-control (2.64 ± 0.22,
2.08 ± 0.15 vs 3.83 ± 0.18 mL) and neutrophils (8.98 ± 1.36, 8.00 ± 0.22 vs 20.51 ± 1.14) in carra-
geenan-induced pouch. EAFMm significantly reduced exudate volume, pro-inflammatory cytokines and the
expression of COX-2 and NFκB.
Conclusion: M. mesozygia leaves demonstrated anti-nociceptive and anti-inflammatory activities by suppressing
oxidative stress, pro-inflammatory cytokines, cyclooxygenase-2, and nuclear factor kappa B.

1. Introduction
Pain is a multidimensional sensory experience, it is intrinsically
afflictive, whose intensity, quality, duration and referral may vary, and
usually it is linked with hurting and soreness (Woolf, 2004). Pain
happens to be one common reason for patients to seek medical atten-
tion and one of the most prevalent medical complaints in the world
(Tompkins et al., 2017). It equally represents important medical and
economic costs for the community (Jagerovic et al., 2002). The devel-
opment and persistence of many pathological pain states has been
linked to inflammatory responses in both the peripheral and central
nervous system (Zhang and An, 2007). The process of nociception and
or pain perception is often described to involve the integration of the
different types of transduction/conduction pathways. The mechanisms
and chemical mediators depend on the nature and site of injury (DeVon
et al., 2014). The pain receptors are widely distributed, and are specific
for mechanical, thermal and toxic chemical or inflammatory mediators
(Bentley et al., 1983). The genesis of pain is inflammatory in most
cases, regardless of the aetiology, thus targeting inflammation and pain
together is an important concept in pain management (DeVon et al.,
2014).
Non-steroidal anti-inflammatory drugs (NSAIDs), opioid analgesics,

∗
Corresponding author.
E-mail addresses: ladekemi2@gmail.com (O.O. Ariyo), am.ajayi@mail1.ui.edu.ng (A.M. Ajayi), pharmben4ver@yahoo.com (B. Ben-Azu),
adebee738@yahoo.co.uk (A.O. Aderibigbe).

https://doi.org/10.1016/j.jep.2020.112934
Received 31 October 2019; Received in revised form 27 April 2020; Accepted 28 April 2020
Available online 07 May 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
O.O. Ariyo, et al.
corticosteroids and analgesic adjuvants such as antidepressants and
local anaesthetics are currently employed in the treatment of in-
flammatory pain. Although their anti-inflammatory and analgesic ac-
tivity cannot be disputed, these agents all have significant side effects
including gastrointestinal and renal damage (Kaushik et al., 2012; Lalan
et al., 2015). This, together with patient dissatisfaction with pain care,
has necessitated the search for novel analgesics and anti-inflammatory
agents, which are more effective in treating inflammation and pain of
any origin and with improved safety profile (Jagerovic et al., 2002). A
great number of people are turning to herbal therapies to find holistic
treatment with effective pain relief and few side effects. Thus, there is a
greater interest in natural compounds from medicinal plants, which
have longstanding successful use in reducing pain and inflammation
(Maroon et al., 2010).
M. mesozygia Stapf (Moraceae) is commonly known as African
mulberry (Dzoyem and Eloff, 2015) or ewe Aiye (Yoruba, Southwest
Nigeria) (Gbile, 1984). The plant can either be a shrub growing up to
6 m tall, or a tree with a very leafy, umbrella-shaped crown, which can
grow from 10 to 40 m tall with a crown spread of 25 m. The roots, stem,
flowers and leaves have been used in folkloric medicine to treat sy-
philis, dermatitis, rheumatism, asthenias, fever, malaria, arthritis,
malnutrition, debility, pain and stomach troubles (Berhaut, 1979;
Burkill et al., 1994). The leaves form part of a mixture of plants used for
treating peptic ulcer by the local people of Bangangte region, of western
Cameroon (Noumi and Dibakto, 2000). The leaves of M. mesozygia is
used ethnomedicine practice of managing different types of arthritis
and several other inflammatory-related disorders in Southern Africa
(Elisha et al., 2016). The literature reports the presence of different
phytochemical compounds in the leaves of Morus species such as phe-
nolic acids, flavonoids, tannins, saponins to which pharmacological
properties have been attributed (Ntie-Kang et al., 2014; Hussain et al.,
2017). The acetone leaf extract of M. mesozygia was previously reported
to reduce nitric oxide production in RAW 264.7 cells as well as pos-
sessing potent anti-oxidant and anticholinesterase activity (Dzoyem and
Eloff, 2015; Elisha et al., 2016). Although previous report has shown
the anti-inflammatory effect of methanol tem bark extract of M. meso-
zygia (Oshiomah et al., 2016), to the best of our knowledge there is no
report showing evidence of the anti-nociceptive and anti-inflammatory
activities and the probable underlying mechanisms of M. mesozygia
leaves extracts. Due to the large variety of compounds found in the
leaves of M. mesozygia, and the need to effectively identify the anti-
inflammatory activity in vivo, this study aimed to evaluate the anti-
nociceptive and anti-inflammatory effects of the ethanol extract, ethy-
lacetate and residual aqueous fractions from M. mesozygia leaves.
2. Materials and methods
2.1. Materials and reagents
The following substances were used: acetic acid (BDH), for-
maldehyde (BDH), morphine (97% purity) and naloxone (99% purity)
(Cristália). Other reagents include carrageenan, indomethacin, O-dia-
nisidine, thiobarbituric acid (TBA), 51, 51-Dithios nitrobenzoic acid
(DTNB) were all products of Sigma Aldrich (Steinheim, Germany).
Biolegend tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6)
ELISA kits (USA). Extraction solvents were re-distilled from commercial
grade solvents.
2.2. Experimental animals
Wistar rats or Swiss mice of either sex were obtained from the
College of Medicine Central Animal House, University of Ibadan. The
animals were, acclimatized in the laboratory for one week before the
commencement of each experiment. They were kept polypropylene
plastic cages lined with wood shaven, under natural ventilation, at
room temperature of 28 ± 2 °C, under natural day and night cycles in
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Journal of Ethnopharmacology 259 (2020) 112934
the experimental laboratory. The rodents were fed on commercial
standard rodents’ chow (Vital feeds Ltd, Jos, Nigeria) and water ad li-
bitum. Experimental procedures were performed with compliance to
The “Principle of Laboratory Animal Care” (NIH Publication No. 85–23)
(National Institute of Health, 1996) and in consideration of ethical re-
quirement for experimental pain in conscious animals (Zimmerman,
1983). The protocols were submitted to and approved by the University
of Ibadan Animal Ethics Committee (UI-ACUREC/App/03/2017/015).
2.3. Plant material and extraction
Leaves of M. mesozygia were collected from the botanical garden at
the University of Ibadan, Nigeria and authenticated by Mr Adeyemi.
Further, http://www.theplantlist.org/was consulted for updated no-
menclature of the plant. Thereafter, herbarium specimen was deposited
in the Forest Research Herbarium with voucher number FHI 110808.
Fresh leaves of M. mesozygia were obtained, rinsed in water and dried
under shade on the laboratory slab. The leaves of the M. mesozygia were
pulverize into coarse powder with the aid of a grinding machine. The
powdered material (800 g) was extracted with aqueous ethanol (50:50,
v/v) for 72 h at room temperature with frequent agitation. The extract
was filtered and the solvent removed under reduced pressure with a
rotary evaporator (Buchi Rotavapor R-124) at 40 °C, and then freeze
dried to obtain crude ethanol extract (EEMm) with a yield of 5.8%. 33 g
of the crude extract (EEMm) was suspended in water and initially
partitioned with n-hexane before repeated partitioning with ethylace-
tate (1:1 v/v) to obtain the ethylacetate (EAFMm) and residual aqueous
fractions (RAFMm). The percentage yield of the EAFMm and RAFMm
were 13.6% and 78.4%, respectively. The extract and fractions were
stored at room temperature under an activated silica gel-desiccator.
Since mechanistic studies involving immunohistochemistry does not
focus on dose-dependent or independent effects, but to see how pre-
viously established effective and non-effective doses act mechan-
istically. Hence, the dose of the extract and fractions used in this pre-
sent study are based on results obtained from our preliminary
investigations and previous studies as well as findings from our acute
toxicity testing of the crude extract at 2000 mg/kg (data not shown).
2.4. Evaluation of anti-nociceptive activity of the ethanol leaves extract and
fractions of M. mesozygia
2.4.1. Hot plate test
The central anti-nociceptive activity of M. mesozygia extract and
fractions was assessed in male Swiss mice, as per the method described
by Eddy and Leimbach (1953). The mice were divided into 8 groups
(n = 5). The treatment group was orally administered different doses
(100 and 400 mg/kg) of the extract and fractions. The positive control
group was treated with morphine (5 mg/kg) intraperitoneally (i.p.) and
the negative control group was administered the vehicle (10 mL/kg; 1%
tween 80). The animals were individually placed on a thermostatically
controlled hot plate (Ugo Basile, Italy) within a restraining perspex
cylinder maintained at 55 ± 1 °C. Reaction time was recorded when
the animals licked their fore-and hind paws or jumped; before (0) and at
60, 90, 120 and 150 min after administration of test drugs. A cut off
time of 20 s was set to avoid tissue damage. The mice that reacted
within 20 s were selected for the study.
The mean percentage maximum possible effect (% MPE) was cal-
culated as:
Post −drug latency  −  Pre−drug latency
% MPE = × 100 
Cut−off time − Post drug latency
Thereafter, to test for involvement of opioidergic system, naloxone
(2 mg/kg, i. p) was injected 15 min prior to administration of the most
active fraction (EAFMm 400 mg/kg, p. o.) or positive control drug
(morphine, 5 mg/kg, i. p.).
O.O. Ariyo, et al.
2.4.2. Acetic acid induced writhing test
The peripheral anti-nociceptive activity of extract and fractions in
mice was evaluated in the model of acetic acid-induced writhing test
(Koster, 1959). Eight groups of mice (n = 5) were orally treated with
100 and 400 mg/kg of either ethanolic extract (EEMm), ethylacetate
fraction (EAFMm) or residual aqueous fraction (RAFMm) 1 h before
intrapeitoneal injection of 10 mL/kg acetic acid (0.6%). The negative
control group was orally treated with 10 mL/kg of vehicle (Tween 80,
1%, v/v) while indomethacin (10 mg/kg) was used as positive control.
The number of acetic acid-induced abdominal writhing response was
counted from 5th to 20th minutes after injection.
2.4.3. Formalin induced licking
Formalin-induced tonic pain was carried out in a similar manner to
the method previously described by Hunskaar and Hole (1987). Eight
groups of mice (n = 5) were orally treated with 100 and 400 mg/kg of
either ethanolic extract (EEMm), ethylacetate fraction (EAFMm) or re-
sidual aqueous fraction (RAFMm). One hour after extract/fraction ad-
ministration by the oral route, each mouse received an intra-plantar
injection of 20 μL of formalin (2.5%) in the right-hind paw and the
duration of paw licking was determined 0–5 min (early phase) and
20–30 min (late phase) after formalin administration. The mice in the
negative control group were treated with 10 mL/kg of vehicle (Tween
80, 1%, v/v) while those in positive control group were orally treated
with indomethacin (10 mg/kg).
2.4.4. Evaluation of the effect of the ethylacetate fraction of M. mesozygia
on mice in the open-field test
This experiment was to assess the possible nonspecific muscle re-
laxant or the locomotor impairment effects of the ethylacetate fraction
(EAFMm) as the most effective fraction, which may mask the motor
performances of mice when tested in the open-field apparatus (Archer,
1973). Five groups of mice (n = 5) were treated with 10 mL/kg of
vehicle (Tween 80, 1%, v/v), 400 mg/kg EAFMm (p.o., for 1 h), 5 mg/
kg morphine (i.p., 30 min) or treated with naloxone (2 mg/kg, i. p.)
15 min prior to administration of EAFMm (400 mg/kg) or morphine
(5 mg/kg, i. p.). The locomotor activity tested was carried using the
UgoBasile activity cage apparatus. Each mouse placed in the cage was
allowed to have free ambulation for 5 min, the number of locomotion
frequency or rearing was recorded from the horizontal beam breaks or
vertical beam breaks.
2.5. Evaluation of the membrane stabilizing activity of ethanol leaves
extract and fractions of M. mesozygia
The membrane stabilizing activity assay was carried out as pre-
viously described by Ajayi et al. (2014). Alsever solution was prepared
by dissolving 2% dextrose, 0.8% Sodium citrate, 0.05% citric acid and
0.42% sodium chloride in distilled water. The resulting solution was
then sterilized in an autoclave. Blood was collected by cardiac puncture
from healthy male Wistar rats. The collected blood was mixed with
equal volume of sterilized alsever solution and centrifuged at 3000 rpm
for 10 min. Packed cells were washed with isosaline (0.85%, pH 7.2)
and a suspension in 10% (v/v) isosaline was prepared.
The membrane stabilizing assay mixture consisted of hyposaline
(2 mL; 0.25% w/v), sodium phosphate buffer at pH 7.4 (1 mL), RBC
suspension (0.3 mL; 10% v/v) and 1 mL of varying concentrations
(0.625, -10 mg/mL) of EEMm, EAFMm, RAFMm and indomethacin. The
assay volume was made up to 5 mL with isosaline solution. The control
was prepared as above except that the drug was omitted, while drug
control lacked erythrocyte suspension. The reaction mixtures were in-
cubated at 56 °C for 30 min in a water bath and the tubes cooled under
running water followed by centrifugation at 4000 rpm for 10 min. The
supernatant was collected followed by reading of the absorbance of
released haemoglobin at 560 nm using a UV/Vis Spectrophotometer
(752N INESA, China). The percentage inhibition of hemolysis or
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Journal of Ethnopharmacology 259 (2020) 112934
membrane stabilization was estimated using the expression:
% Membrane Stability = C-T/C*100
Where, C - Absorbance of Control and T - Absorbance of Test Sample.
2.6. Evaluation of the anti-inflammatory activity of ethanol leaves extract
and fractions of M. mesozygia using the carrageenan-induced rat paw
oedema
Female Wistar rats weighing 150–180 g were divided into eight
groups (n = 5): (i) vehicle (Tween 80, 1%, v/v), (ii) EEMm (100 mg/
kg), (iii) EEMm (400 mg/kg), (iv) EAFMm (100 mg/kg), (v) EAFMm
(400 mg/kg) (vi) RAFMm (100 mg/kg), (vii) RAFMm (400 mg/kg)
(viii) Indomethacin (10 mg/kg). In carrageenan-induced rat paw oe-
dema (Winter et al., 1962), the animals were orally pre-treated for 1 h
before the intraplantar injection of carrageenan (1%, 0.1 mL) into the
right hind foot. There was pre-measurement of paw volumes using Ugo
Basile (7134) digital plethysmometer (Comerio VA, Italy) before car-
rageenan injection and post-carrageenan injection at 1, 2, 3, 4, and 5 h.
The increase in paw volume was calculated as a percentage and plotted
against the experimental time, then the area under the curve for total
oedema (paw volume) was computed (Oshiomah et al., 2016).
The percentage of inhibition of total rat paw oedema was calculated
with the formula
% inhibition = [(AUC of control –AUC of treatment)/AUC of control]
×100%: (Ajayi et al., 2017b )
Five hours after carrageenan injection, the animals were anaes-
thetized with Pentobarbital (50 mg/kg; i. p) and euthanized; the paws
were disaggregated from the joint and stored at -80 °C. Paw tissues were
homogenized using a mechanical grinder and centrifuged at
10,000 rpm for 10 min at 4 °C. Supernatants were then used to assess
myeloperoxidase (MPO) enzyme activity, malondialdehyde (MDA) and
reduced glutathione (GSH) levels.
2.7. Evaluation of the anti-inflammatory mechanisms of the ethylacetate
fraction of M. mesozygia in carrageenan-induced air pouch inflammation
model
Subcutaneous air pouches were formed under anaesthesia according
to the modified method of Martin et al. (1994). Thirty-six female Wistar
rats weighing 180–200 g were anaesthetized with ketamine (50 mg/kg)
and xylazine (5 mg/kg), and the dorsal back was shaven with a sterile
razor, and then a pouch was formed by subcutaneous injection of 20 mL
air. Four days later, another 10 mL of air was injected at the same site
without anaesthetizing the animals. Thereafter, animals were randomly
divided into six groups (n = 6), group 1 (normal control, saline), group
2 (carrageenan-control), group 3 and 4 (EAFMm 100 and 400 mg/
kg + carrageenan), group 5 (Celecoxib 20 mg/kg + carrageenan) and
group 6 indomethacin (5 mg/kg). The groups were pre-treated orally
from day 4–6 as thus; group 1 and 2 received vehicle (Tween 80, 1%, v/
v), group 3 and 4 received 100 and 400 mg/kg of EAFMm, group 5
(Celecoxib 20 mg/kg) while group 6 (indomethacin 5 mg/kg). One hour
after the last pre-treatment, 2 mL carrageenan (1% in sterile saline) was
injected directly into the pouches of rats in groups 2–6 while group 1
received sterile normal saline. 24 h post-carrageenan or saline injection,
the rats were deeply anaesthetized with ether and a small incision was
made into the pouch. This was immediately flushed with 2 mL sterile
phosphate buffered saline (0.1 M, pH 7.4) and fluids recovered mea-
sured. Aliquots of the exudates were separated for total and differential
leucocytes counts. The remaining exudates was separated by cen-
trifugation at 10,000 rpm for 10 min in a refrigerated centrifuge set at
4 °C. The cell free exudate supernatant was aliquoted and stored at
-80 °C for evaluation of inflammatory mediators (TNF-α, IL-6, Nitric
oxide (NO)) and oxidative stress markers (reduced gluthathione (GSH)
O.O. Ariyo, et al.
and malondialdehyde (MDA)). The cell residue was used to assess
myeloperoxidase (MPO) enzyme activity.
2.8. Biochemical analysis
2.8.1. Determination of total protein and nitrite levels
Lavage fluid protein was determined by the Biuret method ac-
cording to the protocol of Gornall et al. (1949) using as bovine serum
albumin as standard. Exudates nitrite level was determined by reacting
with Greiss reagent and absorbance measured at 540 nm using UV–Vis
spectrophotometer (Green et al., 1982).
2.8.2. Determination of pro-inflammatory markers
Cell free pouch fluid aliquots of 100 μL from the air pouch experi-
ment was used to measure the levels of TNFα and IL-6 using ELISA
technique according the protocol described in Biolegend ELISA MAXTM
Deluxe kit manufacturer's manual (Biolegend, USA).
2.8.3. Determination of reduced glutathione levels
The antioxidant marker (GSH) levels in paw tissue supernatants or
cell-free exudate was determined (Sin et al., 1997). The paw tissue
supernatants or cell-free exudate, initially deproteinated with tri-
chloroacetic acid, and thereafter incubated with Ellman's reagent
(DTNB). The absorbance of the reaction mixture was read at 412 nm
and GSH concentration expressed as μM GSH/mg protein or μM GSH/
mL exudates.
2.8.4. Determination of malondialdehyde levels
Lipid peroxides formation in paw tissue supernatants or cell-free
exudate was examined by measuring formation of thiobarbituric-re-
acting substances (TBARS) (Nagababu et al., 2010). Malondialdehyde
(MDA) an end product of lipid peroxidation in tissue was measured at
532 nm absorbance in a UV/Vis spectrophotometer, and expressed as
ƞmol/mg protein or ƞmol/mL exudates.
2.8.5. Determination of myeloperoxidase enzyme activity in paw tissue
homogenates or leucocytes pellets
Paw tissue homogenates or leucocyte pellets were suspended in
assay extraction buffer containing hexadecyltrimethylammonium bro-
mide (0.5%), and 50 Mm potassium phosphate buffer (pH 6.0). The
samples were frozen at -20 °C and undergoes 3 cycles of freeze-thawing
with vigorous shaking intermittently. The final lysed cells were cen-
trifuged at 15,000 rpm in for 15 min in a refrigerated centrifuge set at
4 °C. MPO activity in the supernatant was performed by adding 0.2 mL
of supernatant to 2.8 mL of assay substrate (0.167 mg/mL O-dianisidine
in 50 mM potassium phosphate buffer and 0.15 mM H2O2). The kinetics
of absorbance over 3 min was measured at 450 nm using a UV/VIS
spectrophotometer (INESA). A unit of MPO was defined as that giving a
change in absorbance of 0.001 per min and the specific activity ex-
pressed as unit of MPO per milligram of protein (Bradley, 1982).
2.9. Histopathological and immunohistochemical processing
A cross section of the pouch tissue were cut and fixed in 10% neutral
buffered formalin. Sections were processed through paraffin-embedded
in paraffin blocks. A sectioning of 4 μm was used for hematoxylin and
eosin (H&E) staining and observed for histopathological changes. The
tissues were also stained immunohistochemically for cyclooxygenase-2
(COX-2) and nuclear factor kappa B (NF-κB) expression using biorbyt
polyclonal antibodies (Cambridge, UK). The tissues were processed
with 4% paraformaldehyde, with subsequent washing in hydrogen
peroxide in methanol solution (0.3%) to quench peroxidase. Tissue
slices were incubated with goat antisera in 0.6% Triton X-100 in PBS.
The tissue slices were incubated at 4 °C overnight with rabbit polyclonal
antibody against COX-2 or NF-κB p65 (biorbyt, UK). Following 24 h of
incubation, the tissues were washed with PBS and incubated with rabbit
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Journal of Ethnopharmacology 259 (2020) 112934
anti-goat IgG antibody (HRP) (biorbyt, UK). Subsequently, the slides
were processed by incubation in a one-step horseradish peroxidase
(HRP) and after repeated washing developed with 3, 3′- diamino-
benzidine (DAB) reagent. Thereafter, slides were incubated with he-
matoxylin and Images captured to a computer interface (MagnaFire)
through a camera mounted on Olympus BX-51 Binocular research mi-
croscope. The photomicrographs of immunopositive cells were used for
quantitative analysis of expression with the aid Image J software (NIH,
Bethesda, MD, USA).
2.10. Phytochemical fingerprinting of the M. mesozygia leaf ethylacatate
fraction by high performance liquid chromatography (HPLC)
Preliminary phytochemical fingerprinting of the ethylacetate frac-
tion was performed by HPLC technique (Ajayi et al., 2017b). The unit is
a Shimadzu HPLC system with SIL- 20AC auto-sampler, a DGU-20A3
degasser component and column oven CTO-20AC. Other components
include SPD-M20A UV-diode array detector that works with a system
controller CBM- 20Alite and Windows LC solution software (Shimadzu
Corporation, Kyoto Japan). The sample injection volume was 5 μL of
200 μg/mL solution of the ethylacetate fraction dissolved in methanol
as the mobile phase. The mobile phase consisted of: solvent A: 0.2% v/v
formic acid; solvent B: acetonitrile; mode The technique used an iso-
cratic elution at a flow rate 0.6 mL/min. The detection was set at UV
254 nm wavelength. The available reference standards (gallic acid,
rutin, quercetin, caffeic acid, ferulic acid and morin) were also used to
identify some likely compounds in the fraction.
2.11. Data analysis
Data are expressed as Mean ± SEM. Statistical significance is taken
for p < 0.05, and analysis was by one-way analysis of variance
(ANOVA) followed by Newman Keuls test using the GraphPad Prism
version 5.
3. Results
3.1. Anti-nociceptive activity of M. mesozygia leaves ethanol extract and
fractions in the hot plate test
The EEMm (100 and 400 mg/kg), EAFMm (100 mg/kg) and RAF
(100 and 400 mg/kg) did not significantly prolong reaction time when
compared to control mice (Fig. 1A&B). Only the EAFMm (400 mg/kg)
showed effectiveness in the hot plate test by significantly increasing the
maximal possible effect when compared to the control mice. Morphine
(5 mg/kg) showed a significant (p < 0.001) maximal possible effect
when compared to control. As shown in Fig. 1C and D, pre-treatment
with naloxone (1 mg/kg) did not reversed the anti-nociceptive effect of
EAFMm (400 mg/kg) but blocked the anti-nociceptive activity of
morphine (5 mg/kg).
3.2. Anti-nociceptive activity of M. mesozygia leaves ethanol extract and
fractions in the acetic acid-induced writhing test
The crude extract and its fractions showed a significant and dose
dependent anti-nociceptive effect in the acetic acid-induced writhing in
mice (Fig. 2). The maximum inhibition of writhing was recorded for the
EAFMm (400 mg/kg) at 66.5%, which compared favourably against
standard (indomethacin, 10 mg/kg) with 66.3% inhibition.
3.3. Anti-nociceptive activity of M. mesozygia leaves ethanol extract and
fractions in the formalin test
The EEMm, EAFMm and RAFMm at the lower dose (100 mg/kg) did
not significantly reduce the duration of paw-licking and biting after
formalin injection in the early phase (0 – 5min) compared to the control
O.O. Ariyo, et al.
Fig. 2. Anti-nociceptive activity of ethanolic extract (EEMm), ethylacetate
fraction (EAFMm) and residual aqueous fraction (RAFMm) of M. mesozygia
leaves in the acetic acid-induced writhing test. Data represents Mean ± SEM of
five mice. *p < 0.05, **p < 0.01, ***p < 0.001 vs control (1-way ANOVA
followed by Newman-Keuls Multiple comparison post hoc test).
(Fig. 3A). At 400 mg/kg however, EEMm and EAFMm produced a
significant (p < 0.05) decrease in the duration of paw-licking at the
early phase (Fig. 3B). During the late phase (20–30 min), EEMm (100
and 400 mg/kg), EAFMm (100 mg/kg) and RAFMm (100 and 400 mg/
kg) significantly (p < 0.05) reduced duration of paw-licking and biting
after formalin injection (Fig. 3C). Indomethacin (10 mg/kg) showed a
significant (p < 0.05) reduction in paw-licking and biting in both
5
Journal of Ethnopharmacology 259 (2020) 112934
Fig. 1. Anti-nociceptive activity of ethanolic extract
(EEMm), ethylacetate fraction (EAFMm) and residual
aqueous fraction (RAFMm) of M. mesozygia leaves in
the hot plate test. (A) Maximal possible effect of ex-
tract/fractions (B) Area under the curve (AUC) of
extract/fractions, (C) Maximal possible effect in
presence of naloxone (D) AUC in the presence of
naloxone. Data represents Mean ± SEM of five
mice.**p < 0.01, ***p < 0.001 vs control; **p <
0.01, #p < 0.01 vs morphine (1-way ANOVA fol-
lowed by Newman-Keuls Multiple comparison post
hoc test).
phases when compared with the control.
3.4. Locomotory and exploratory activity
In the open field test, the EAFMm (100 mg/kg) and Morphine
(5 mg/kg) had no significant effect on locomotor activity (horizontal
activity count) compared with the control (Fig. 4A). Pre-treatment with
naloxone (2 mg/kg) did not significantly reverse the effect of EAFMm
(400 mg/kg) or morphine (5 mg/kg) on locomotory activity (Fig. 4A).
On exploratory activity or rearing (vertical activity count), EAFMm
400 mg/kg had no significant effect compared to control. However, the
exploratory activity was significantly (p < 0.05) reduced on pre-
treatment with naloxone. Morphine (5 mg/kg) significantly reduced
exploratory activity in the open field and on prior administration of
naloxone, there was no reversal of the activity (Fig. 4B).
3.5. Anti-inflammatory activity of M. mesozygia leaves ethanol extract and
fractions
3.5.1. Membrane stabilizing activity
The varying concentrations (0.125, 0.25, 0.5, 1.0 and 2.0 mg/mL) of
the crude extract (EEMm), ethylacetate fraction (EAFMm) and residual
aqueous fraction (RAFMm) showed dose-dependent membrane stabi-
lizing activity (Fig. 5A). The inhibitory concentration (IC50) ranked as
follows; EEMm (IC50 0.38 ± 0.06 mg/mL) > EAFMm (IC50
0.49 ± 0.03 mg/mL) > RAFMm (IC50 0.52 ± 0.04 mg/mL). The
standard drug, indomethacin showed a more potent membrane
Fig. 3. Anti-nociceptive activity of ethanolic extract
(EEMm), ethylacetate fraction (EAFMm) and residual
aqueous fraction (RAFMm) of M. mesozygia leaves in
the formalin test. (A) Early phase (B) Late phase.
Data represents Mean ± SEM of five mice. *p <
0.05, **p < 0.01, ***p < 0.001 vs control (1-way
ANOVA followed by Newman-Keuls Multiple com-
parison post hoc test).
O.O. Ariyo, et al. Journal of Ethnopharmacology 259 (2020) 112934

Fig. 4. Effect of ethylacetate fraction (EAFMm

400 mg/kg) and morphine on locomotory activity in
the open field test. (A) Horizontal count (Locomotory
activity) and (B) Vertical count (exploratory ac-
tivity). Data represents Mean ± SEM of five mice.
*p < 0.05, vs control, *p < 0.05 vs EAFMm
400 mg/kg (1-way ANOVA followed by Newman-
Keuls Multiple comparison post hoc test).

stabilizing activity with IC50 of 0.32 ± 0.04 mg/mL (Fig. 5B).

3.5.2. Antioedema effects in carrageenan-induced rat paw oedema

Carrageenan caused a progressive swelling in the paw of rats from
the 0th to the 5th hour in the carrageenan control group (Fig. 6A). The
interpolation of the percentage increase in paw volume as area under
the curve (AUC) showed that the ethanol extract and its fractions sig-
nificantly (p < 0.001) decreased paw volume (Fig. 6B). The EEMm
(100 and 400 mg/kg) inhibited by 36.1% and 43.3% the increase in
paw oedema, while EAFMm (100 and 400 mg/kg) reduced in 53.4%
and 58.1% and RAFMm reduced in 29.4% and 45.7%, respectively
(Fig. 6B). The positive control group, indomethacin significantly
(p < 0.001) reduced the paw volume by 59.0% compared to the
control.

3.5.3. Effect of M. mesozygia leaves ethanol extract and fractions on

carrageenan-induced rat paw myeloperoxidase, malondialdehye and
reduced glutathione levels
The myeloperoxidase enzyme activity as shown in Fig. 7A was
significantly reduced by pre-treatment of rats with doses of ethanol
extract, ethylacetate and residual aqueous fractions of M. mesozygia
Fig. 6. Anti-inflammatory activity of ethanolic extract (EEMm), ethylacetate
leaves. At the 400 mg/kg, EEMm, EAFMm and RAFMm decreased MPO fraction (EAFMm) and residual aqueous fraction (RAFMm) of M. mesozygia
activity to a greater extent when compared to that of indomethacin leaves in the carrageenan-induced rat paw oedema. (A) Percentage increase in
(10 mg/kg). The crude extract and fractions at 400 mg/kg significantly paw volume (B) Area under the curve (AUC). Data represents Mean ± SEM of
(p < 0.05) decreased the levels of malondialdehyde (MDA) in the five rats. **p < 0.01, ***p < 0.001 vs control (1-way ANOVA followed by
carrageenan-induced paws compared with the control (Fig. 7B). In- Newman-Keuls Multiple comparison post hoc test).
domethacin (10 mg/kg) also reduced MDA expression significantly but
to a lesser degree to the effect observed with EEMm (400 mg/kg), 400 mg/kg) and RAFMm (400 mg/kg), whereas the EEMm and RAFMm
EAFMm (100 and 400 mg/kg) and RAFMm (400 mg/kg). did not prevent GSH depletion in the carrageenan-induced rat paw.
Fig. 7C shows that the level of reduced glutathione (GSH) was sig- Indomethacin also did not show significant protection against
nificantly elevated by pre-treatment of rats with EAFMm (100 and

Fig. 5. Membrane stabilizing activity of ethanolic extract (EEMm), ethylacetate fraction (EAFMm) and residual aqueous fraction (RAFMm) of M. mesozygia leaves on
rat erythrocytes. (A) % inhibition of hemolysis, (B) IC50. Values are in triplicate readings.

6
O.O. Ariyo, et al.
carrageenan-induced GSH depletion in the rat paws.
3.6. Anti-inflammatory activity of ethylacetate fraction of M. Mesogyzia
(EAFMm) on carrageenan-induced air pouch in rat
3.6.1. EAFMm reduces the volume of exudates, protein concentration,
malondialdehyde and elevates GSH
Carrageenan in a 6-day old air pouch caused a significant
(p < 0.0001) increase in exudate formation and accumulation of
protein into the cavity in 24 h. Fluid exudation in the carrageenan-
induced air pouch was reduced by EAFMm (100 and 400 mg/kg), cel-
ecoxib (20 mg/kg) and indomethacin (10 mg/kg), significantly
(Fig. 8A). A significant (p < 0.05) decline in exudate total protein
compared to the carrageenan control was also observed for EAFMm
(100 and 400 mg/kg) at 35.2% and 44.5% inhibition respectively.
Likewise, Celecoxib (20 mg/kg) and Indomethacin (10 mg/kg) sig-
nificantly (p < 0.05) reduced exudate total protein by 31.7% and
36.4% respectively (Fig. 8B). The anti-oxidant effects of pre-treatment
with EAFMm were assessed by determining MDA and GSH levels in the
exudates. The result indicates that the presence of carrageenan in the
7
Journal of Ethnopharmacology 259 (2020) 112934
Fig. 7. Effect of ethanolic extract (EEMm), ethyla-
cetate fraction (EAFMm) and residual aqueous frac-
tion (RAFMm) of M. mesozygia leaves on in-
flammatory and oxidative stress markers in the
carrageenan-induced rat paw. (A) myeloperoxidase
activity, (B) malondialdehyde levels and (C) reduced
glutathione levels. Data represents Mean ± SEM of
five rats. *p < 0.05, **p < 0.01, ***p < 0.001 vs
control (1-way ANOVA followed by Newman-Keuls
Multiple comparison post hoc test).
pouches increased the levels of malondialdehyde (MDA) in the carra-
geenan-induced air-pouch compared to the control. EAFMm (100 and
400 mg/kg) was able to reduce the index of lipid peroxidation in the
carrageenan-injected pouch (Fig. 8C). Treatment with EAFMm (100 and
400 mg/kg) prevented glutathione depletion (Fig. 8D).
3.6.2. EAFMm reduces the inflammatory cells migration into air pouch
Carrageenan in a 6-day old air pouch caused a significant increase in
leucocytes migration (Fig. 9A), predominantly of neutrophils (Fig. 9B).
Treatment with 100 and 400 mg/kg EAFMm showed inhibition of ac-
cumulation of total leucocytes and the differential inflammatory cells
(neutrophils, monocytes and lymphocytes; Fig. 9A–D) in the air pouch.
The percentage inhibition of total leucocytes migration by EAFMm (100
and 400 mg/kg) were 49.0% and 57.5%; for neutrophils 56.2% and
61.0%; monocytes 61.0% and 55.9%; lymphocytes 31.4% and 46.6%
respectively. Celecoxib (20 mg/kg) and indomethacin (10 mg/kg) de-
monstrated comparable inhibition of total leucocytes 48.3% and 51.8%;
neutrophils 56.4% and 60.7%; monocytes 44.6% and 67.8%; lympho-
cytes 31.1% and 33.4% respectively.
Fig. 8. Effect of ethylacetate fraction (EAFMm) of M.
mesozygia leaves in carrageenan-induced pouch exu-
dates (A) exudates volume, (B) protein concentration
(C) malondialdehyde, and (D) reduced glutathione.
Data represents Mean ± SEM of five rats.
#p < 0.05 vs sal or *p < 0.05, **p < 0.01,
***p < 0.001 vs CGN (1-way ANOVA followed by
Newman-Keuls Multiple comparison post hoc test).
Sal – normal saline, CGN-carrageenan-control, EAF-
ethylacetate fraction.
O.O. Ariyo, et al. Journal of Ethnopharmacology 259 (2020) 112934

Fig. 9. Effect of ethylacetate fraction (EAFMm) of M.

mesozygia leaves on inflammatory cells accumulation
in carrageenan-induced pouch exudates (A) Total
leucocytes count, (B) neutrophils (C) monocytes, and
(D) lymphocytes. Data represents Mean ± SEM of
five rats. #p < 0.05 vs sal or *p < 0.05, **p <
0.01, ***p < 0.001 vs CGN (1-way ANOVA fol-
lowed by Newman-Keuls Multiple comparison post
hoc test). Sal – normal saline, CGN-carrageenan-
control, EAF- ethylacetate fraction.

3.6.3. EAFMm reduces tumor necrosis factor-α (TNF-α), interleukin-6 (IL- EAFMm (100 and 400 mg/kg) pre-treated animals, there was a reduc-
6) levels, nitrite levels and the index of neutrophil activation tion in MPO enzyme activity by 49.5% and 57.1% respectively
(myeloperoxidase MPO activity) (Fig. 10D). At the same time, the control groups treated with celecoxib
Carrageenan in a 6-day old air pouch caused the elevation of pro- (20 mg/kg) and indomethacin (10 mg/kg) produced inhibitions of
inflammatory cytokines and nitric oxide. Treatment of rats with 100 58.2% and 60.5% respectively.
and 400 mg/kg EAFMm significantly reduced TNF-α levels. The per-
centage of inhibition was calculated to be 76.0% and 62.7% respec-
3.6.4. Effect of EAFMm on carrageenan-induced histopathological
tively.
alteration of the air-pouch tissue
A similar effect was observed with IL-6 (Fig. 10B), with EAFMm
Carrageenan in a 6-day old air pouch caused enlargement of pouch
(100 and 400 mg/kg) significantly inhibiting IL-6 generation by 56.4%
walls and infiltration of mononuclear inflammatory cells (lymphocytes
and 64.4% respectively. Likewise, nitrite (Fig. 10C) was significantly
and monocytes) and neutrophils. Inflammatory cells are absent in the
reduced by 44.0% in EAFMm 100 mg/kg and 63.3% in EAFMm
saline group. Pretreatment with EAFMm (100, 400 mg/kg) and
(400 mg/kg)-treated rats. Celecoxib (20 mg/kg) and Indomethacin
Celecoxib (20 mg/kg) suppressed the number of infiltrating leukocytes
(10 mg/kg) significantly (p < 0.05) reduced both TNF-α (71.0% and
as sparse amounts of mononuclear inflammatory cells and neutrophils
47.0%) and nitrite (23.7% and 31.2%) levels in the exudates respec-
were observed compared to the carrageenan group. Moderate amounts
tively. Inflammation caused by carrageenan in the air-pouch is linked
of mononuclear inflammatory cells just below the epidermis and
with infiltration of neutrophils into the pouch tissue lining. In the
around degenerate hair follicles was observed in the indomethacin

Fig. 10. Effect of ethylacetate fraction (EAFMm) of

M. mesozygia leaves on inflammatory mediators in
carrageenan-induced pouch exudates (A) TNF-α, (B)
IL-6 (C) nitrites, and (D) MPO. Data represents
Mean ± SEM of five rats. #p < 0.05 vs sal or *p <
0.05, **p < 0.01, ***p < 0.001 vs CGN (1-way
ANOVA followed by Newman-Keuls Multiple com-
parison post hoc test). Sal – normal saline, CGN-
carrageenan-control, EAF- ethylacetate fraction.

8
O.O. Ariyo, et al. Journal of Ethnopharmacology 259 (2020) 112934

Fig. 11. Representative photomicrographs of histopathological examination of the carrageenan-induced pouch linning (hematoxylin-eosin, x 400).

(10 mg/kg) group (Fig. 11). 3.7. High performance liquid chromatographic analysis of the ethylacetate
fraction of Morus mesozygia leaf

3.6.5. Effect of EAFMm on carrageenan-induced COX-2 and nuclear factor HPLC fingerprinting of EAFMm resolved a total of sixteen peaks
kappa B (NF-κB) expressions in the pouch of rats detected at a typical UV absorption wavelength of 254 nm (Fig. 14). Six
There is high expression of cyclooxygenase-2 (COX-2) in the car- phenolic compounds were identified by the peaks representing gallic
rageenan group (Fig. 12B) which was reduced in the EAFMm 400 mg/ acid, caffeic acid, rutin, ferulic acid, morin and quercetin in the ethy-
kg, celecoxib and indomethacin groups (Fig. 12E–F). EAFMm 100 mg/ lacetate fraction.
kg did not significantly suppress COX-2 expression in the rats’ pouch.
Quantification of percent immunopositive for COX-2 showed sig-
4. Discussion
nificantly reduced area in groups treated with EAFMm (400 mg/kg),
celecoxib and indomethacin (Fig. 12 G). Similarly, photomicrograph
This study reported the anti-nociceptive and anti-inflammatory ac-
expression of NF-κB was higher in carrageenan control group compared
tivities of ethanol extract and fractions of M. mesozygia leaves and the
to saline (Fig. 13A–B). The quantification of immunopositive area for
mechanism of anti-inflammation of the ethylacetate fraction in ex-
NF-κB in animals treated with EAFMm (100 and 400 mg/kg) showed
perimental models.
significant reduced levels of NF-κB. Celecoxib and indomethacin sig-
The anti-nociceptive effect of the ethanolic extract and ethylacetate
nificantly reduced NF-κB expression in the pouch tissues (Fig. 13G).
and residual aqueous fractions of M. mesozygia leaves was examined in
the hot plate test. The EAFMm (400 mg/kg) only showed effectiveness

Fig. 12. Representative photomicrograph of

immunohistochemical staining for COX-2 in
the carrageenan-induced air pouch tissue.
(A) Normal saline, (B) Carrageenan (CGN),
(C) EAFMm 100 mg/kg, (D) EAFMm
400 mg/kg, (E) Celecoxib 20 mg/kg, and (F)
Indomethacin 10 mg/kg. (G) Quantitative
analysis of COX-2 positive cells. Data re-
presents Mean ± SEM of three samples.
#p < 0.05 vs sal or *p < 0.05, **p <
0.01, ***p < 0.001 vs CGN (1-way ANOVA
followed by Newman-Keuls Multiple com-
parison post hoc test). Sal – normal saline,
CGN-carrageenan-control, EAF- ethylace-
tate fraction.

9
O.O. Ariyo, et al.
Fig. 14. Phytochemical profile of ethylacetate fraction analyzed by HPLC.
in the hot plate model, as it significantly increased the reaction time to
pain when compared to the control mice. Pretreatment with naloxone
did not reverse the analgesic effect produced by EAFMm (400 mg/kg).
Morphine showed a significant maximal possible effect when compared
to control, an effect which was reversed by pre-treatment with na-
loxone. This observation suggests that the central analgesic effect of the
ethylacetate fraction might not be related to the activation of opioid
receptors mediating central analgesic effect.
The acetic acid induced writhing model for the study of pain pri-
marily assesses peripherally acting analgesics. Acetic acid causes pain
by liberating endogenous substances such as serotonin, histamine,
bradykinins, substance P and prostaglandins (PGs) in the peritoneal
cavity (Deraedt et al., 1980; Bentley et al., 1983). The prostaglandins
are involved in lowering the threshold to stimulation of nociceptors in
the pain pathway and promptly inhibited by the NSAIDs (Ghaisas et al.,
2010). In the present study, the crude extract and fractions of M. me-
sozygia exhibited significant inhibition of writhing response induced by
acetic acid hence suggesting the involvement of peripheral mechanisms
of analgesia.
An important model in screening anti-nociceptive activity is the
10
Journal of Ethnopharmacology 259 (2020) 112934
Fig. 13. Representative photomicrograph of
immunohistochemical staining for NF-κB in
the carrageenan-induced air pouch tissue.
(A) Normal saline, (B) Carrageenan (CGN),
(C) EAFMm 100 mg/kg, (D) EAFMm
400 mg/kg, (E) Celecoxib 20 mg/kg, and (F)
Indomethacin 10 mg/kg. (G) Quantitative
analysis of NF-κB positive cells. Data re-
presents Mean ± SEM of three samples.
#p < 0.05 vs sal or *p < 0.05, **p <
0.01, ***p < 0.001 vs CGN (1-way ANOVA
followed by Newman-Keuls Multiple com-
parison post hoc test). Sal – normal saline,
CGN-carrageenan-control, EAF- ethylace-
tate fraction.
formalin-induced nociception, which considered to be biphasic, con-
sisting of the neurogenic early phase and the inflammatory late phase.
The inflammatory second phase is characterized by a slowly developing
inflammatory pain that is as a result of release of prostaglandins, his-
tamine, serotonin, bradykinin and cytokines (IL-1β, IL-6, TNF-α)
(Pinho-Ribeiro et al., 2017). Formalin develops a hyperalgesic state in
the late phase of the test (Chichorro et al., 2004). The first phase is
inhibited by opioid analgesics while the late phase is inhibited by non-
steroidal anti-inflammatory drugs and opioid analgesics (Kaufmann
et al., 1997). The analgesia observed in the early phase by EEMm
(400 mg/kg), EAFMm (400 mg/kg) and indomethacin is probably a
result of membrane stabilizing action of these agents. The crude extract
and fractions significantly inhibited nociception in the late phase (in-
flammatory pain). The greater pain suppression at the late phase than
the early phase could be due to the combined effect of inhibition of the
synthesis of prostaglandins and other inflammatory cytokines/media-
tors and membrane stabilization activities of the extract and fractions.
The pain suppression at the second phase is suggestive of anti-
hyperalgesic and peripheral activity and hints on the possibility that the
extract contains agents that exhibit anti-inflammatory property.
O.O. Ariyo, et al.
NSAIDs have been reported to reduce pain at both the phases of the
formalin test when acting supraspinally (Dharmasiri et al., 2003; Kumar
et al., 2013), which was observed with indomethacin (10 mg/kg) as
well as EEMm (400 mg/kg) and EAFMm (400 mg/kg).
The effect of EAFMm that had shown pronounced anti-nociceptive
activity was further evaluated on the spontaneous locomotor activity of
mice in order to rule out any false positive effect. EAFMm did not
change the locomotor activity and exploratory rearing activity in mice
unlike morphine that significantly reduced the rearing activity.
However, pre-treatment with naloxone prior to EAFmm significantly
depressed the rearing activity in mice. Naloxone has been known to
depress rearing activity in mice (Czech et al., 1983). The constituents in
the ethylacetate fraction might be acting at different opioid sites fa-
cilitating and as well suppressing the μ-opioid receptor (Ayoka et al.,
2013). The results revealed that the pronounced anti-nociceptive effect
of EAFMm was unconnected to any inhibition of locomotor activity in
mice. The analgesic effect exhibited by the extract and fractions re-
sembled the effect of anti-inflammatory agents to a large extent and to
lesser extent the centrally acting analgesic drugs.
The ethanol extract, ethylacetate and residual aqueous fractions of
M. mesozygia showed variable membrane stabilizing activity against
heat-induced erythrocytes haemolysis. The inflammatory process is
accompanied by the release of lysosomal enzymes, such as proteases,
into the cytosol causing tissue damage and resulting into the develop-
ment of a variety of disorders (Sadique et al., 1989). The erythrocyte
membrane is similar to lysosomal membrane thus, the effect of drugs on
the stabilization of erythrocyte membrane could be likened/extra-
polated to the stabilization of lysosomal membranes (Omale and
Okafor, 2008). Reports have shown that non-steroidal anti-in-
flammatory agents act by stabilizing lysosomal membranes or in-
activating already released enzymes and these have been utilized as an
adjunctive model in the screening and study of anti-inflammatory drugs
or extracts in vitro (Kumar and Sadique, 1987; Sonibare et al., 2015).
The ethanolic extract and fractions of M. mesozygia leaves demon-
strated anti-inflammatory activity in carrageenan-induced rat paw oe-
dema. Carrageenan-induced paw oedema is an acute inflammatory
model for detecting orally active anti-inflammatory agents (Lamien
et al., 2006). Oedema formation in the rat paw is biphasic involving
vascular and inflammatory mediators. The first phase (0–1 h), mediated
through the release of histamine, serotonin and bradykinin, is not
blocked by the use of NSAIDs such as indomethacin or aspirin. The
second phase (1–6 h) is the outcome of elevated levels of prostaglandins
and COX-2 in tissue macrophages (Di Rosa et al., 1971; Vinegar et al.,
1987). Pre-treatment of rats with extract and fractions of M. mesozygia
leaves significantly reduced the paw oedema in both phases, which
demonstrated the anti-oedematogenic activity of M. mesozygia leaves.
The suppression of inflammation is likely the result of the inhibition of
prostaglandins and kinin synthesis/release. The ethylacetate fraction
showed the most profound anti-oedema activity similar to in-
domethacin, this is in consonance with the reported antiinflammatory
properties of NSAIDs and some extracts (Suleyman et al., 2004; Ajayi
et al., 2017a).
Acute inflammation is characterized by activation of cells and re-
lease of inflammatory mediators, such as histamine, serotonin and
prostaglandins that work jointly to heighten vasodilatation and per-
meability of blood vessels. This leads to increased blood flow, exudation
of plasma proteins and fluids, and migration of leukocytes, mainly
neutrophils, outside the blood vessels into the injured tissues.
Myeloperoxidase (MPO) is an enzyme found in neutrophils. MPO ac-
tivity is an indication of neutrophil infiltration and activation, and it
directly linked with chemotactic activity at inflammatory site in the
tissue (Smith, 1994). It is possible that extract and fractions inhibits
activation of neutrophils as shown by the reduction in paw tissue MPO
enzyme activity.
The protective effect of M. mesozygia extract and fractions was
further shown in the levels of malondialdehyde (MDA) and GSH.
11
Journal of Ethnopharmacology 259 (2020) 112934
Malondialdehyde production is utilized as a biomarker to evaluate
oxidative stress level in an organism (Del Rio et al., 2005). The second
phase of carrageenan-induced rat paw oedema has been linked with
free radical related lipid peroxidation (Ajayi et al., 2017a). Thus, ex-
tracts inhibiting the increase in neutrophilic infiltration and activation
are shown to reduce the index of lipid peroxidation (Mazzon et al.,
2001). The extract and fractions of M. mesozygia exhibited a significant
reduction in MDA activity especially at the higher doses. This indicates
the beneficial effect of M. mesozygia on lipid peroxidation stimulated by
oxidants on the polyunsaturated fatty acids of the membranes (Riedel
et al., 2015). EAFMm and RAFMm 400 mg/kg exhibited a significant
dose dependent elevation of reduced glutathione levels. Reports have
shown that GSH metabolism is critically essential to many biological
processes, especially in neutralizing xenobiotic reaction of oxygen
species and free radicals (Sin et al., 1997). Evaluation of oxidative stress
during a free-radical challenge is considered a good predictor anti-in-
flammatory processes in experimental conditions for therapeutic mea-
sures (Sarithakumari et al., 2013). The in vitro anti-oxidant activity of
the acetone leaf extract of M. mesozygia was recently reported (Elisha
et al., 2016). The ability of the ethylacetate fraction to depress MDA
activity and raise GSH levels suggests that it probably possesses anti-
oxidant activity.
To further give credence to the observed anti-inflammatory activity
of M. mesozygia leaf ethylacetate fraction, the air-pouch model was
employed. The ethylacetate fraction of M. mesozygia suppressed the
inflammatory response in a 6-day old carrageenan-induced air-pouch.
Phlogistic agents in the air-pouch triggers an inflammatory response
typified by infiltration of immune cells (Sedgwick and Lees, 1986), and
because it provides a synovial-like milieu, it is suitable for studying
acute inflammatory outcomes and the mode of action of anti-in-
flammatory agents (Martin et al., 1994; Romano et al., 1997; Eteraf-
Oskouei et al., 2015; Duarte et al., 2012). A significant increase in
exudate volume and total leucocyte count, including neutrophils,
monocytes and lymphocytes, was observed in the rat air-pouches in-
jected with carrageenan in the group administered saline only. Pre-
treatment with the ethylacetate fraction of M. mesozygia suppressed the
fluid exudation and the number of leukocytic cells in a dose dependent
manner. Also reflected in the data is the reduction of the levels of MDA
and elevation of GSH levels in the inflammatory exudates of EAFMm-
treated rats. The level of glutathione as reported, is normally elevated
in response to carrageenan injection, however, it gradually becomes
depleted when it reacts with free radicals to yield thiol products (Sin
et al., 1997); as observed in the air-pouch exudate of the animals in-
jected carrageenan. Pretreatment with EAFMm was able to reverse this
trend.
Tumor necrosis factor –alpha level in the carrageenan-exudates was
significantly elevated than in the saline-fluids, which was significantly
reduced in animals treated with EAFMm. This might probably be by
regulating the local output of TNF-α in the carrageenan-induced air
pouch inflammation. TNF-α plays an important role as an inflammatory
mediator in pathogenesis of rheumatoid arthritis, regulates leucocyte
migration in the acute inflammatory phase, activates neutrophils by
promoting leakage into the lungs, liver, gut and other organs. Spewed
neutrophils harm tissues lining due to the release of oxygen-free radi-
cals and proteases (Romano et al., 1997; Liz et al., 2011; Li and Ng,
2012). In the same manner, the levels of IL-6, MPO and nitrite were
significantly reduced in the inflammatory exudates of EAFMm treated
rats. Neutrophils generate and release these mediators (Silva et al.,
2015), hence, their decreased levels could be a function of a decline in
the neutrophil count in the exudates of the EAFMm treated rats. A si-
milar trend was observed with proteins; there was a decrease in the
level of proteins in the exudates of EAFMm treated rats compared with
control. For comparison, pretreatment with celecoxib and in-
domethacin suppressed exudate volume, leucocyte counts, MDA levels
and elevated glutathione levels. They were also able to reverse carra-
geenan-induced elevation of TNF-α, IL-6, MPO, nitrite and protein
O.O. Ariyo, et al.
levels in the exudates. The reduction in the level of nitric oxide was
probably the result of an inhibition of inducible nitric oxide synthase
(iNOS) expression. iNOS is expressed mainly as a response to certain
inflammatory stimuli such as bacterial products, cytokines and lipid
mediators (Moncada et al., 1976; Laroux et al., 2001) and it generates
colossal quantities of nitric oxide for prolonged periods in response to
inflammatory or pro-inflammatory mediators (Laroux et al., 2001). In
addition, histological investigation of the pouch tissue lining injected
carrageenan showed profound blood cells infiltration, which was re-
duced in the pouch lining of EAFMm-treated rats. This observation is
consistent with other studies where a heightened infiltration of blood
cells in carrageenan injected pouch tissue of rats suggests ongoing in-
flammatory reaction and when such infiltration is reduced, an anti-in-
flammatory reaction has taken place (Anilkumar et al., 2017).
A drop in MPO activity is suggestive of the inhibition of the mi-
gration and activation of neutrophils. Reports have shown poly-
morphonuclear cells represents a larger percentage of the total leuko-
cytes in carrageenan-induced acute inflammation (Sin et al., 1997).
Neutrophil activation is associated with radical species generation and
degranulation. The presence of activated neutrophils in synovial fluids
of patients with rheumatoid arthritis is linked with inflammatory pro-
gression and joint destruction and characterized by elevated release of
neutrophilic reactive oxygen species (Nurcombe et al., 1991). This
further confirms the anti-inflammatory ability of EAFMm in restraining
leucocytes migration and especially neutrophils mobilization and acti-
vation at inflammatory sites. Pro-inflammatory cytokines such as TNF-α
and IL-6 are released during an inflammatory response; their partici-
pation is controlled by transcription factors, particularly nuclear factor
kappa B (NFκB) (Rai et al., 2018). This explains the surge in the exudate
levels of these cytokines, and increased expression of NFκB positive
cells in the pouch tissues on carrageenan injection. Thus, inhibition of
NFκB expression will translate to reduction in the production of the pro-
inflammatory cytokines and subsequent inhibition of inflammation.
COX-2 is one of the fundamental mediators of inflammation whose
expression in air-pouch tissues is heightened by carrageenan injection
(Yu et al., 2016; Anilkumar et al., 2017), but was downregulated by
administration of EAFMm in the present study.
5. Conclusion
Taken together, the findings above demonstrate that M. mesozygia
leaves ethanolic extract, ethylacetate and residual aqueous fractions
possess promising anti-nociceptive and anti-inflammatory effects. The
superior efficacy of the ethylacetate fraction might be due to a con-
centration of the active compounds acting synergistically to improve
the anti-nociceptive and anti-inflammatory activities. The strength of
the present study was that M. mesozygia leaves ethanolic extract,
ethylacetate and residual aqueous fractions reduces nociceptive and
inflammatory response in rodents in different animal models by in-
hibition of pro-inflammatory enzymes, cytokines, COX-2 expression
through NF-κB activation.
Contributions
OOA, AMA, AAA designed the work, and OOA, AMA, BB performed
the experiments. All of the authors analyzed and interpreted the data.
OOA. and AMA develop the draft manuscript, and all of the other au-
thors contributed to editing and approved the final manuscript.
Acknowledgements
Authors thank Mr Tosin of the Department of Pharmaceutical
Chemistry for his assistance in extraction and partitioning. We also
appreciate Mr Olatunde Matthews of the Department of Pharmacology
and Therapeutics for technical assistance during the experiments.
12
Journal of Ethnopharmacology 259 (2020) 112934
References
Ajayi, A.M., Martins, D.T.O., Balogun, S.O., Oliveira, R.G., Ascencio, S.D., Soares, I.M.,
Barbosa, R.B., Ademowo, O.G., 2017a. Ocimum gratissimum L. leaf flavonoid-rich
fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages
and peritonitis in mice. J. Ethnopharmacol. 204, 169–178.
Ajayi, A.M., Tanayen, J.K., Ezeonwumelu, J.O.C., Dare, S., Okwanachi, A., Adzu, B.,
Ademowo, O.G., 2014. Anti-inflammatory, anti-nociceptive and total polyphenolic
content of hydroethanolic extract of Ocimum gratissimum L. leaves. Afr. J. Med. Med.
Sci. 43s, 215.
Ajayi, A.M., Umukoro, S., Ben‐Azu, B., Adzu, B., Ademowo, O.G., 2017b. Toxicity and
protective effect of phenolic‐enriched ethylacetate fraction of Ocimum gratissimum
(linn.) leaf against acute inflammation and oxidative stress in rats. Drug Dev. Res. 78,
135–145.
Anilkumar, K., Reddy, G.V., Azad, R., Yarla, N.S., Dharmapuri, G., Srivastava, A., Kamal,
M.A., Pallu, R., 2017. Evaluation of anti-inflammatory properties of isoorientin iso-
lated from tubers of Pueraria tuberosa. Oxid. Med. Cell. Longev. 2017.
Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim. Behav. 21,
205–235.
Ayoka, A.O., Owolabi, R.A., Bamitale, S.K., Akomolafe, R.O., Aladesanmi, J.A.,
Ukponmwan, E.O., 2013. Effect of fractionated extracts and isolated pure compounds
of spondias mombin (L. Anacardiaceae) leaves on novelty-induced rearing and
grooming behaviors in mice. Afr. J. Tradit., Complementary Altern. Med. 10,
244–255.
Bentley, G.A., Newton, S.H., Starr, J., 1983. Studies on the antinociceptive action of
α‐agonist drugs and their interactions with opioid mechanisms. Br. J. Pharmacol. 79,
125–134.
Berhaut, J., 1979. Flore illustrée du Sénégal, Dicotylédones, Linacées à Nymphéacées,
Tome VI. In: Dakar: Gouvernement du Sénégal, Ministère du Développement Rural et
de lʼHydraulique. Direction des eaux et Forêts, pp. 466–481.
Bradley, P.P., 1982. Christensen RD, Rothstein G. Cellular and extracellular myeloper-
oxidase in pyogenic inflammation. Blood 60, 618–622.
Burkill, H.M., Families, E.I., 1994. The Useful Plants of West Tropical Africa, vol. 2 Royal
Botanic Gardens.
Chichorro, J.G., Lorenzetti, B.B., Zampronio, A.R., 2004. Involvement of bradykinin,
cytokines, sympathetic amines and prostaglandins in formalin‐induced orofacial
nociception in rats. Br. J. Pharmacol. 141, 1175–1184.
Czech, D.A., Stein, E.A., Blake, M.J., 1983. Naloxone-induced hypodipsia: a CNS mapping
study. Life Sci. 33, 797–803.
Del Rio, D., Stewart, A.J., Pellegrini, N., 2005. A review of recent studies on mal-
ondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr.
Metabol. Cardiovasc. Dis. 15, 316–328.
Deraedt, R., Jouquey, S., Delevallée, F., Flahaut, M., 1980. Release of prostaglandins E
and F in an algogenic reaction and its inhibition. Eur. J. Pharmacol. 61, 17–24.
DeVon, H.A., Piano, M.R., Rosenfeld, A.G., Hoppensteadt, D.A., 2014. The association of
pain with protein inflammatory biomarkers: a review of the literature. Nurs. Res. 63,
51–62.
Dharmasiri, M.G., Jayakody, J.R.A.C., Galhena, G., Liyanage, S.S.P., Ratnasooriya, W.D.,
2003. Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex
negundo. J. Ethnopharmacol. 87, 199–206.
Di Rosa, M.L., Giroud, J.P., Willoughby, D.A., 1971. Studies of the mediators of the acute
inflammatory response induced in rats in different sites by carrageenan and tur-
pentine. J. Pathol. 104, 15–29.
Duarte, D.B., Vasko, M.R., Fehrenbacher, J.C., 2012. Models of inflammation: carra-
geenan air pouch. Curr. Protoc. Pharmacol. 56, 5–6.
Dzoyem, J.P., Eloff, J.N., 2015. Anti-inflammatory, anticholinesterase and antioxidant
activity of leaf extracts of twelve plants used traditionally to alleviate pain and in-
flammation in South Africa. J. Ethnopharmacol. 160, 194–201.
Eddy, N.B., Leimbach, D., 1953. Systematic analgesics II. Diethyl butenyl and diethie-
nylbutyl amines. J. Pharmacol. Exp. Therapeut. 107, 387–393.
Elisha, I.L., Dzoyem, J.P., McGaw, L.J., Botha, F.S., Eloff, J.N., 2016. The anti-arthritic,
anti-inflammatory, antioxidant activity and relationships with total phenolics and
total flavonoids of nine South African plants used traditionally to treat arthritis. BMC
Compl. Alternative Med. 16, 307.
Eteraf-Oskouei, T., Allahyari, S., Akbarzadeh-Atashkhosrow, A., Delazar, A., Pashaii, M.,
Gan, S.H., Najafi, M., 2015. Methanolic Extract of Ficus Carica Linn. Leaves Exerts
Antiangiogenesis Effects Based on the Rat Air Pouch Model of Inflammation.
Evidence-Based Complementary And Alternative Medicine 2015.
Gbile, Z.O., 1984. Vernacular Names of Nigerian Plants (Yoruba). ” Forestry Research
Institute of Nigeria, Ibadan.
Ghaisas, M.M., Dandawate, P.R., Zawar, S.A., Ahire, Y.S., Gandhi, S.P., 2010.
Antioxidant, antinociceptive and anti-inflammatory activities of atorvastatin and
rosuvastatin in various experimental models. Inflammopharmacology 18, 169–177.
Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum protein by
means of biuret reaction. J. Biol. Chem. 177, 751.
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R.,
1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal.
Biochem. 126 (1), 131–138.
Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between in-
flammatory and non-inflammatory pain. Pain 30, 103–114.
Hussain, F., Rana, Z., Shafique, H., Malik, A., Hussain, Z., 2017. Phytopharmacological
potential of different species of Morus alba and their bioactive phytochemicals: a
review. Asian Pac. J. Trop. Biomed. 7, 950–956.
Jagerovic, N., Cano, C., Elguero, J., Goya, P., Callado, L.F., Meana, J.J., Giron, R., Abalo,
R., Ruiz, D., Goicoechea, C., 2002. Long-acting fentanyl analogues: synthesis and
O.O. Ariyo, et al.
pharmacology of N-(1-phenylpyrazolyl)-N-(1-phenylalkyl-4-piperidyl) propana-
mides. Bioorg. Med. Chem. 10, 817–827.
Kaufmann, W.E., Andreasson, K.I., Isakson, P.C., Worley, P.F., 1997. Cyclooxygenases and
the central nervous system. Prostaglandins 54, 601–624.
Kaushik, D., Kumar, A., Kaushik, P., Rana, A.C., 2012. Analgesic and anti-inflammatory
activity of Pinus roxburghii sarg. Adv. Pharmacol. Sci. 2012.
Koster, R., 1959. Acetic acid for analgesic screening. Fed Proc. 18, 412.
Kumar, P.S., Sadique, J., 1987. The biochemical mode of action of Gynandropsis gy-
nandra in inflammation. Fitoterapia 58, 379–386.
Kumar, S.S., Marella, S.S., Vipin, S., Sharmistha, M., 2013. Evaluation of analgesic and
anti-inflammatory activity of Abutilon indicum. Int. J. Drug. Develop. Res. 5,
1402–1407.
Lalan, M., Baweja, J., Misra, A., 2015. Atopic dermatitis: drug delivery approaches in
disease management. Crit. Rev. Ther. Drug Carrier Syst. 32, 323–361.
Lamien, C.E., Guissou, I.P., Nacoulma, O.G., 2006. Anti-inflammatory, analgesie and
antipyretic activities of Dicliptera verticillata. Int. J. Pharmacol. 2, 435–438.
Laroux, F.S., Pavlick, K.P., Hines, I.N., Kawachi, S., Harada, H., Bharwani, S., Hoffman,
J.M., Grisham, M.B., 2001. Role of nitric oxide in inflammation. Acta Physiol. Scand.
173, 113–118.
Li, J.L., Ng, L.G., 2012. Peeking into the secret life of neutrophils. Immunol. Res. 53,
168–181.
Liz, R., Pereira, D.F., Horst, H., Dalmarco, E.M., Dalmarco, J.B., Simionatto, E.L.,
Pizzolatti, M.G., Girard, D., Fröde, T.S., 2011. Protected effect of Esenbeckia leio-
carpa upon the inflammatory response induced by carrageenan in a murine air pouch
model. Int. Immunopharm. 11, 1991–1999.
Maroon, J.C., Bost, J.W., Maroon, A., 2010. Natural anti-inflammatory agents for pain
relief. Surg. Neurol. Int. 1, 80.
Martin, S.W., Stevens, A.J., Brennan, B.S., Davies, D., Rowland, M., Houston, J.B., 1994.
The six-day-old rat air pouch model of inflammation: characterization of the in-
flammatory response to carrageenan. J. Pharmacol. Toxicol. Methods 32, 139–147.
Mazzon, E., Serraino, I., Li, J.H., Dugo, L., Caputi, A.P., Zhang, J., Cuzzocrea, S., 2001.
GPI 6150, a poly (ADP-ribose) polymerase inhibitor, exhibits an anti-inflammatory
effect in rat models of inflammation. Eur. J. Pharmacol. 415, 85–94.
Moncada, S., Gryglewski, R.J., Bunting, S., Vane, J.R., 1976. An enzyme isolated from
arteries transforms prostaglandin endoperoxides to an unstable substance that in-
hibits platelet aggregation. Nature 263, 663.
Nagababu, E., Rifkind, J.M., Boindala, S., Nakka, L., 2010. Assessment of antioxidant
activity of eugenol in vitro and in vivo. Free Radic. Antioxid. Protoc. 2010, 165–180.
National Institute of Health, 1996. Institute of Laboratory Animal Resources (US).
Committee on Care and Use of Laboratory Animals, 1986. Guide For the Care and Use
of Laboratory Animals. US Department of Health and Human Services, Public Health
Service.
Noumi, E., Dibakto, T.W., 2000. Medicinal plants used for peptic ulcer in the Bangangte
region, western Cameroon. Fitoterapia 71, 406–412.
Ntie-Kang, F., Onguéné, P.A., Lifongo, L.L., Ndom, J.C., Sippl, W., Mbaze, L.M.A., 2014.
The potential of anti-malarial compounds derived from African medicinal plants, part
II: a pharmacological evaluation of non-alkaloids and non-terpenoids. Malar. J.
13, 81.
Nurcombe, H.L., Bucknall, R.C., Edwards, S.W., 1991. Neutrophils isolated from the sy-
novial fluid of patients with rheumatoid arthritis: priming and activation in vivo.
Ann. Rheum. Dis. 50, 147–153.
Omale, J., Okafor, P.N., 2008. Comparative antioxidant capacity, membrane stabilization,
polyphenol composition and cytotoxicity of the leaf and stem of Cissus multistriata.
Afr. J. Biotechnol. 7, 17.
13
Journal of Ethnopharmacology 259 (2020) 112934
Oshiomah, K.O., Odujoko, O.A., Ajayi, A.M., 2016. Anti-inflammatory effects and acute
toxicity of methanol stem bark extract of Morus mesozygiaStapf. Br. J. Pharmaceut.
Res. 13, 1–9.
Pinho-Ribeiro, F.A., Verri Jr., W.A., Chiu, I.M., 2017. Nociceptor sensory neuron-immune
interactions in pain and inflammation. Trends Immunol. 38, 5–19.
Rai, U., Rawal, A., Singh, S., 2018. Evaluation of the anti-inflammatory effect of an anti-
platelet agent crinumin on carrageenan-induced paw oedema and granuloma tissue
formation in rats. Inflammopharmacology 26, 769–778.
Riedel, R., Marrassini, C., Anesini, C., Gorzalczany, S., 2015. Anti‐inflammatory and
antinociceptive activity of urera aurantiaca. Phytother Res. 29, 59–66.
Romano, M., Sironi, M., Toniatti, C., Polentarutti, N., Fruscella, P., Ghezzi, P., Faggioni,
R., Luini, W., Van Hinsbergh, V., Sozzani, S., Bussolino, F., 1997. Role of IL-6 and its
soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 6,
315–325.
Sadique, J., Al-Rqobahs, W.A., Bughaith, E.I., Gindi, A.R., 1989. The bioactivity of certain
medicinal plants on the stabilization of RBC membrane system. Fitoterapia 60,
525–532.
Sarithakumari, C.H., Renju, G.L., Kurup, G.M., 2013. Anti-inflammatory and antioxidant
potential of alginic acid isolated from the marine algae, Sargassum wightii on ad-
juvant-induced arthritic rats. Inflammopharmacology 21, 261–268.
Sedgwick, A.D., Lees, P., 1986. Studies of eicosanoid production in the air pouch model of
synovial inflammation. Agents Actions 18, 429–438.
Silva, A.M.D.O.E., Machado, I.D., Santin, J.R., de Melo, I.L.P., Pedrosa, G.V., Genovese,
M.I., Farsky, S.H.P., Mancini‐Filho, J., 2015. Aqueous extract of Rosmarinus offici-
nalis L. inhibits neutrophil influx and cytokine secretion. Phytother Res. 29, 125–133.
Sin, Y.M., Pook, S.H., Tan, T.M., Petterssoon, A., Kara, A.U., The, W.F., 1997. Changes in
gluthathione and its associated enzymes during carrageenan-induced acute in-
flammation in mice. Comp. Biochem. Physiol. 191–195.
Smith, J.A., 1994. Neutrophils, host defense, and inflammation: a double‐edged sword. J.
Leukoc. Biol. 56, 672–686.
Sonibare, M.A., Onda, E.E., Ajayi, A.M., Umukoro, S., 2015. In vitro antioxidant and
membrane stabilization activities of the fruit extract and fractions of Tetrapleura
tetraptera (Schumach & Thonn.) Taub. J. Pharm. Bioresour. 12, 95–105.
Suleyman, H., Demircan, B., Karagoz, Y., Oztasan, N., Suleyman, B., 2004. Anti-in-
flammatory effects of selective COX-2 inhibitors. Pharmacol. Rep. 56, 775–780.
Tompkins, D.A., Hobelmann, J.G., Compton, P., 2017. Providing chronic pain manage-
ment in the "Fifth Vital Sign" Era: historical and treatment perspectives on a modern-
day medical dilemma. Drug Alcohol Depend. 173 (Suppl. 1), S11–S21.
Vinegar, R., Truax, J.F., Selph, J.L., Johnston, P.R., Venable, A.L., McKenzie, K.K., 1987.
January. Pathway to carrageenan-induced inflammation in the hind limb of the rat.
Fed. Proc. 46, 118–126.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind paw of
the rat as an assay for antiinflammatory drugs. Exp. Biol. Med. 111, 544–547.
Woolf, C.J., 2004. Pain: moving from symptom control toward mechanism-specific
pharmacologic management. Ann. Intern. Med. 140, 441–451.
Yu, Y., Li, X., Qu, L., Chen, Y., Dai, Y., Wang, M., Zou, W., 2016. DXXK exerts anti-
inflammatory effects by inhibiting the lipopolysaccharide-induced NF-κB/COX-2
signalling pathway and the expression of inflammatory mediators. J.
Ethnopharmacol. 178, 199–208.
Zhang, J.M., An, J., 2007. Cytokines, inflammation and pain. Int. Anesthesiol. Clin.
45, 27.
Zimmerman, M., 1983. Committee for Research and Ethical Issues of the IASP, Ethical
standards for investigations of experimental pain in animals. Pain 16, 109–110.