anti-inflammatory and antinociceptive activities of homalium letestui
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2013
http://informahealthcare.com/phbISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. PezzutoPharm Biol, 2013; 51(11): 1459–1466
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.799707
ORIGINAL ARTICLE
Anti-inflammatory and antinociceptive activities of Homalium letestui
Jude E. Okokon1, Patience J. Okokon1, Ahsana Dar Farooq2, and Mohammed Iqbal Choudhary2
1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria and 2International Center for Chemical and
Biological Sciences, University of Karachi, Karachi, Pakistan
Abstract
Context: Homalium letestui Pellegr (Flacourtiaceae) is used in various decoctions traditionally bythe Ibibios of the Niger Delta of Nigeria to treat stomach ulcer, malaria and other inflammatorydiseases, as well as an aphrodisiac.Objective: To investigate the anti-inflammatory and antinociceptive activities of the stem extractof the plant.Materials and methods: The ethanol stem extract (500, 750, 1000 mg/kg, i.p.) of H. letestui wasinvestigated for anti-inflammatory activity using carrageenan, egg albumin-induced andxylene-induced ear edema models and analgesic activity using acetic acid-induced writhing,formalin-induced paw licking and thermal-induced pain models. The ethanol extract wasadministered to the animals orally, 30 min to 1 h depending on the model, before induction ofinflammation/pain. The LD50 was also determined. GC–MS analysis of dichloromethane fractionwas carried out.Results: The extract caused a significant (p50.05–0.001) reduction of inflammation induced bycarrageenan (8.3–70.0%), egg albumin (10.0–71.42%) and xylene (39.39–84.84%). The extractalso reduced significantly (p50.05–0.001) pain induced by acetic acid (44.22–73.65%), formalin(55.89–79.21%) and hot plate (93.0–214.5%). The LD50 was determined to be 4.38� 35.72 g/kg.Discussion and conclusion: The results of this study suggest that the ethanol stem extract ofH. letestui possesses anti-inflammatory and analgesic properties which may in part be mediatedthrough the chemical constituents of the plant as revealed by the GC–MS.
Keywords
Analgesic, anti-angiogenic, Flacourtiaceae,phytochemicals
History
Received 6 February 2013Revised 9 April 2013Accepted 23 April 2013Published online 17 July 2013
Introduction
Homalium letestui Pellegr (Flacourtiaceae) is a forest tree
growing up to 80–100 ft and found in the rainforest of West
Africa (Hutchinson & Daziel, 1963; Keay, 1989). The plant
parts, particularly stem bark and root, are used in various
decoctions traditionally by the Ibibios of the Niger Delta of
Nigeria to treat stomach ulcer, malaria and other inflamma-
tory diseases as well as an aphrodisiac (Okokon et al., 2006).
Reports of antiplasmodial (Okokon et al., 2006), antidiabetic
(Okokon et al., 2007), cellular antioxidant, anticancer and
antileishmanial (Okokon et al., 2013) activities of the plant
have been published. However, other members of the genus
Homalium have been reported to possess various biological
activities; Homalium deplanchei Warburg (Flacourtiaceae)
has antileishmanial, antitrypanosomal and antitrichomonal
activities (Desrivot et al., 2007), Homalium panayanum F.
Villar (Flacourtiaceae) has been reported to exert antibacterial
activity against some Gram-positive and Gram-negative
bacteria (Chung et al., 2004), Homalium cochinchinensis
(Lour) Druce (Salicaceae) has antiviral activity (Ishikawa
et al., 2004), Homalium africanum (Hook. F) (Flacourtiaceae)
has filaricidal activity (Cho-Ngwa et al., 2010) and anthel-
mintic activity has been reported on Homalium zeylanicum
(Gardner) Benth (Flacourtiaceae) (Gnananath et al., 2012).
Information on the pharmacology and phytochemistry of
H. letestui is scarce. We report in this study the anti-
inflammatory and antinociceptive activities of this plant to
provide scientific basis for its use in traditional medicine in
treating inflammatory diseases.
Materials and methods
Plants collection
The plant material H. letestui (stem) was collected in a forest
in Uruan area, Akwa Ibom State, Nigeria, in April 2011. The
plant was identified and authenticated by Dr. Margaret Bassey
of Department of Botany and Ecological Studies, University
of Uyo, Uyo, Nigeria. Herbarium specimen (FPUU 382) was
deposited at Department of Pharmacognosy and Natural
Medicine Herbarium.
Extraction
The stem was washed and shade-dried for two weeks. The
dried plant material was further chopped into small pieces and
reduced to powder. The powdered material was macerated in
Correspondence: Jude E. Okokon, Department of Pharmacology andToxicology, Faculty of Pharmacy, University of Uyo, #2 IKPA Road,Uyo 52001, Nigeria. Tel: +234-8023453678. E-mail: judeefiom@yahoo.com
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70% ethanol. The liquid filtrates were concentrated and
evaporated to dryness in vacuo at 40 �C using a rotary
evaporator. The crude ethanol extract (100 g) was further
partitioned successively into 1 L each of n-hexane, dichlor-
omethane, ethyl acetate and butanol to give the corresponding
fractions of these solvents.
Animals
Albino Wistar rats (175–185 g) of either sex were obtained
from the University of Uyo animal house. They were
maintained on standard animal pellets and water ad libitum.
Permission and approval for animal studies were obtained
from the College of Health Sciences Animal Ethics commit-
tee, University of Uyo.
Determination of median lethal dose (LD50)
The median lethal dose (LD50) of the ethanol extract was
estimated in albino mice using the method of Miller and
Tainter (1944). This involved intraperitoneal administration
of different doses of the extract (1000–5000 mg/kg) to groups
of six mice each. The animals were observed for manifest-
ation of physical signs of toxicity such as writhing, decreased
motor activity, decreased body/limb tone, decreased respir-
ation and death.
Evaluation of anti-inflammatory activity of the extract
Carrageenan-induced mice hind paw edema
Adult albino mice of either sex were used for the study. They
were fasted for 24 h and deprived of water only during the
experiment. Inflammation of the hind paw was induced by
injection of 0.1 ml of freshly prepared carrageenan suspension
in normal saline into the subplanar surface of the hind paw.
The linear circumference of the injected paw was measured
before and 0.5, 1, 2, 3, 4 and 5 h after administration of
phlogistic agent. The increase in paw circumference post
administration of phlogistic agent was adopted as the
parameter for measuring inflammation (Akah & Nwanbie,
1994; Besra et al., 1996; Ekpendu et al., 1994; Nwafor et al.,
2010; Winter et al., 1962). The difference in paw circumfer-
ence between the control and 0.5, 1, 2, 3, 4 and 5 h after
administration of phlogistic agent was used to assess inflam-
mation (Hess & Milonig, 1972). The extract (500, 750 and
1000 mg/kg i.p.) was administered to various groups of mice,
1 h before inducing inflammation. Control mice received
carrageenan while reference group received acetyl salicylic
acid (ASA) (100 mg/kg). The average (mean) edema was
assessed by measuring with vernier calipers. The percentage
of inhibition of edema volume between treated and control
groups were calculated using the following formula:
Inhibition %¼ 100� (Vc – Vt)/Vc, where Vc and Vt repre-
sent the mean increases in paw volume in the control and
treated groups, respectively.
Egg albumin-induced inflammation
Inflammation was induced in mice by the injection of egg
albumin (0.1 ml, 1% in normal saline) into the subplanar
tissue of the right hind paw (Akah & Nwanbie, 1994; Okokon
& Nwafor, 2010). The linear circumference of the injected
paw was measured before and 0.5, 1, 2, 3, 4 and 5 h after
the administration of the phlogistic agent. The stem extract
(500, 750 and 1000 mg/kg i.p.) and ASA (100 mg/kg p.o.)
were administered to 24 h fasted mice 1 h before the induction
of inflammation. The control group received 10 ml/kg of
distilled water orally. Edema (inflammation) was assessed as
the difference in paw circumference between the control and
0.5, 1, 2, 3, 4 and 5 h post administration of the phlogistic
agent (Hess & Milonig, 1972). The average (mean) edema
was assessed by measuring with vernier calipers. The
percentage of inhibition of edema volume between treated
and control groups were calculated using the following
formula: Inhibition %¼ 100� (Vc�Vt)/Vc, where Vc and
Vt represent the mean increases in paw volume in the control
and treated groups, respectively.
Xylene-induced ear edema
Inflammation was induced in mice by topical administration
of two drops of xylene at the inner surface of the right ear.
The xylene was left to act for 15 min. H. letestui stem extract
(500, 750 and 1000 mg/kg i.p.), dexamethasone (4 mg/kg) and
distilled water (0.2 ml/kg) were orally administered to various
groups of mice 30 min before the induction of inflammation.
The animals were sacrificed under light anesthesia and the
left ears cut-off. The difference between the ear weights was
taken as the edema induced by the xylene (Mbagwu et al.,
2007; Okokon & Nwafor, 2010; Tjolsen et al., 1992).
Evaluation of analgesic potential of the extract
Acetic acid-induced writhing in mice
Writhing (abdominal constrictions consisting of the contrac-
tion of abdominal muscles together with the stretching of hind
limbs), resulting from intraperitoneal (i.p.) injection of 3%
acetic acid, was induced according to the procedure described
by Santos et al. (1994), Correa et al. (1996) and Nwafor et al.
(2010). The animals were divided into five groups of six mice
per group. Group 1 served as negative control and received
10 ml/kg of normal saline, while groups 2, 3 and 4 were pre-
treated with 500, 750 and 1000 mg/kg doses of H. letestui
extract intraperitoneally, and group 5 received 100 mg/kg
of acetyl salicylic acid. After 30 min, 0.2 ml of 2% acetic
acid was administered intraperitoneally (i.p.). The number of
writhing movements was counted for 30 min. Antinociception
(analgesia) was expressed as the reduction of the number of
abdominal constrictions between control animals and mice
pretreated with extracts.
Formalin-induced hind paw licking in mice
A procedure similar to that described by Hunskaar and Hole
(1987), Correa and Calixto (1993), Gorski et al. (1993) and
Okokon and Nwafor (2010) was adopted for the study. The
animals were injected with 20 ml of 2.5% formalin solution
(0.9% formaldehyde) made up in phosphate buffer solution
(PBS concentration: NaCl 137 mM, KCl 2.7 mM and phos-
phate buffer 10 mM) under the surface of the right hind paw.
The amount of time spent licking the injected paw was timed
and considered as indication of pain. Adult albino mice
(20–25 g) of either sex randomized into five groups of six
1460 J. E. Okokon et al. Pharm Biol, 2013; 51(11): 1459–1466
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mice each were used for the experiment. The mice used were
fasted for 24 h before the experiment but allowed access to
water. The animals in group 1 (negative control) received
10 ml/kg of normal saline, groups 2–4 received 500, 750 and
1000 mg/kg doses of the extract, while group 5 received
100 mg/kg of acetyl salicylic acid (ASA) 30 min before being
challenged with buffered formalin. The responses were
measured for 30 min (first and second phase) after formalin
injection.
Thermally induced pain in mice
The effect of extract on hot plate-induced pain was investigated
in adult mice. The hot plate was used to measure the response
latencies according to the method of Vaz et al. (1996) and
Okokon and Nwafor (2010). In this experiment, the hot plate
was maintained at 45� 1 �C, each animal was placed into a
glass beaker of 50 cm diameter on the heated surface, and the
time(s) between placement and shaking or licking of the paws
or jumping was recorded as the index of response latency. An
automatic 30 sec cut-off was used to prevent tissue damage.
The animals were randomly divided into five groups of six
mice each and fasted for 24 h but allowed access to water.
Group 1 animal served as negative control and received 10 ml/
kg of normal saline. Groups 2, 3 and 4 were pretreated
intraperitoneally with 500, 750 and 1000 mg/kg doses of H.
letestui extract, respectively, while group 5 animals received
100 mg/kg of acetyl salicylic acid intraperitoneally, 30 min
prior to the placement on the hot plate.
GC–MS analysis of dichloromethane fraction
Quantitative and qualitative data were determined by GC and
GC–MS, respectively. The fraction was injected onto a
Shimadzu GC-17A system, equipped with an AOC-20i
autosampler and a split/splitless injector. The column used
was a DB-5 (Optima-5), 30 m, 0.25 mm i.d., 0.25mm df, coated
with 5% diphenyl-95% polydimethylsiloxane, operated with the
following oven temperature program: 50 �C, held for 1 min,
rising at 3 �C/min to 250 �C, held for 5 min, rising at 2 �C/min
to 280 �C, held for 3 min; injection temperature and volume,
250 �C and 1.0ml, respectively; injection mode, split; split ratio,
30:1; carrier gas, nitrogen at 30 cm/s linear velocity and inlet
pressure 99.8 KPa; detector temperature, 280 �C; hydrogen,
flow rate, 50 ml/min; air flow rate, 400 ml/min; make-up (H2/
air), flow rate, 50 ml/min; sampling rate, 40 ms. Data were
acquired by means of GC solution software (Shimadzu).
Agilent 6890N GC was interfaced with a VG analytical
70–250 s double-focusing mass spectrometer. Helium was
used as the carrier gas. The MS operating conditions were
ionization voltage 70 eV, ion source 250 �C. The GC was
fitted with a 30 m� 0.32 mm fused capillary silica column
coated with DB-5. The GC operating parameters were
identical with those of GC analysis described above.
The identification of components present in the various
active fractions of the plant extracts was based on direct
comparison of the retention times and mass spectral data
with those for standard compounds, and by computer
matching with the Wiley and Nist Library, as well as by
comparison of the fragmentation patterns of the mass
spectra with those reported in the literature (Adams, 2001;
Setzer et al., 2007).
Statistical analysis and data evaluation
Data obtained from this work were analyzed statistically using
Student’s t-test and ANOVA (One-way) followed by a post
test (Tukey–Kramer multiple comparison test). Differences
between means were considered significant at 1% and 5%
level of significance, that is, p� 0.01 and 0.05.
Results
Determination of median lethal dose (LD50)
The median lethal dose (LD50) was calculated to be
4.38� 35.72 g/kg. The physical signs of toxicity included
excitation, paw licking, increased respiratory rate, decreased
motor activity, gasping and coma which was followed by
death.
Carrageenan-induced edema in mice
The effect of ethanol stem extract of H. letestui on
carrageenan-induced edema is shown in Table 1. The extract
(500–1000 mg/kg) exerted a significant (p50.05–0.001) anti-
inflammatory effect which was comparable to the standard
drug, ASA (100 mg/kg). The percentage reduction of inflam-
mation was 8.3–70.0% (Table 1).
Egg albumin-induced edema
Administration of stem extract of H. letestui (500–1000 mg/
kg) caused a significant (p50.05–0.001) anti-inflammatory
effect against edema caused by egg albumin in mice with a
Table 1. Effect of Homalium letestui stem extract on carrageenan-induced edema in mice.
Time intervals (h)
Treatment/dose(mg/kg) 0 0.5 1 2 3 4 5
Control 0.23� 0.01 0.35� 0.01 0.36� 0.01 0.35� 0.01 0.34� 0.01 0.33� 0.01 0.31� 0.01Extract500 0.22� 0.01 0.33� 0.01 (8.3) 0.35� 0.01a (0.0) 0.32� 0.01b (16.6) 0.30� 0.01b (27.2) 0.28� 0.01b (60.0) 0.27� 0.01b (16.6)750 0.24� 0.01 0.34� 0.01 (16.6) 0.34� 0.01a (23.0) 0.31� 0.01b (41.6) 0.29� 0.01b (54.5) 0.27� 0.01b (70.0) 0.26� 0.01b (66.6)1000 0.23� 0.01 0.34� 0.01 (8.3) 0.33� 0.01b (23.0) 0.30� 0.01b (41.7) 0.28� 0.01b (54.5) 0.26� 0.01b (70.0) 0.25� 0.01b (66.6)ASA 100 0.24� 0.01 0.34� 0.01 (16.6) 0.34� 0.01a (23.0) 0.32� 0.01b (33.3) 0.30� 0.01b (45.5) 0.28� 0.01b (60.0) 0.25� 0.01b (83.3)
Data are expressed as mean� SEM. Significant at ap50.05, bp50.001 when compared to control. n¼ 6. Values in parentheses represent % inhibitionof inflammation.
DOI: 10.3109/13880209.2013.799707 Biological activities of Homalium letestui 1461
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considerable % reduction of inflammation (10.0–71.42%).
The effect was comparable to that of standard drug, ASA
(100 mg/kg) (Table 2).
Xylene-induced ear edema
Anti-inflammatory effect of stem extract of H. letestui against
xylene-induced ear edema in mice is shown in Table 3.
The extract exerted pronounced anti-inflammatory effect which
was significant (p50.01) with a prominent reduction of
inflammation (39.39–84.84%) and comparable to that of the
standard drug, dexamethasone (4.0 mg/kg) at the highest dose
(1000 mg/kg).
Effect of ethanol crude extract of stem of H. letestui onacetic acid-induced writhing in mice
The administration of H. letestui extract (500–1000 mg/kg)
demonstrated a considerable reduction in acetic acid-induced
writhing in mice with percentage reductions range of 44.22–
73.65%. The reductions were statistically significant (p50.001)
relative to control and comparable to that of the standard drug,
ASA, at the highest dose, 1000 mg/kg (Table 4).
Effect of ethanol stem extract of H. letestui onformalin-induced hind paw licking in mice
The stem extract exhibited a prominent effect on formalin-
induced hind paw licking in mice with percentage inhibition
range of 55.89 to 79.21%. This inhibition was significant
relative to the control (p50.001) and comparable to that of the
standard drug, ASA, at the highest dose, 1000 mg/kg (Table 5).
Effect of ethanol crude extract of stem of H. letestui onthermally induced pain in mice
The stem extract (500–1000 mg/kg) exhibited a consider-
able effect on thermally induced pain in mice. This
inhibition was statistically significant (p50.001) relative
to the control (Table 6). The percentage inhibition range
was 93.0–214.5%.
GC–MS analysis
The results of GC–MS analysis of dichloromethane fraction
of stem extract of H. letestui revealed the presence of
pharmacologically active compounds (Table 7).
Table 2. Effect of Homalium letestui stem extract on egg albumin-induced edema in mice.
Time intervals (h)
Treatment/dose(mg/kg) 0 0.5 1 2 3 4 5
Control 0.24� 0.01 0.32� 0.01 0.34� 0.01 0.34� 0.01 0.33� 0.01 0.32� 0.01 0.31� 0.01Extract500 0.25� 0.01 0.34� 0.01 0.34� 0.01 (10.0) 0.33� 0.01 (20.2) 0.31� 0.01a (22.2) 0.29� 0.01a (50.0) 0.28� 0.01a (57.4)750 0.25� 0.01 0.33� 0.01 0.33� 0.01 (20.0) 0.32� 0.01a (30.0) 0.29� 0.01a (55.5) 0.29� 0.01b (50.0) 0.27� 0.01b (71.4)1000 0.23� 0.01 0.33� 0.01 0.33� 0.01 (0.0) 0.31� 0.01a (20.0) 0.28� 0.01b (44.4) 0.27� 0.01b (50.0) 0.25� 0.01b (71.4)ASA 100 0.24� 0.01 0.35� 0.01a 0.34� 0.01a (0.0) 0.32� 0.01b (20.0) 0.26� 0.01b (77.7) 0.26� 0.01b (75.0) 0.25� 0.01b (85.7)
Data are expressed as mean� SEM. Significant at ap50.01, bp50.001 when compared to control. n¼ 6. Values in parentheses represent % inhibitionof inflammation.
Table 4. Effect of Homalium letestui stem extract on acetic acid-induced writhing in mice.
Time intervals (min)
Treatment/dose (mg/kg) 5 10 15 20 25 30 Total % Reduction
Control 6.00� 1.21 10.33� 1.38 17.20� 1.42 19.14� 1.16 16.51� 0.62 13.24� 0.95 82.32� 6.74Extract 0.00c 8.26� 0.73 10.01� 1.18a 9.30� 1.06c 10.11� 1.20c 8.23� 0.72c 45.91� 4.09c 44.22500750 0.00c 6.44� 0.80 9.10� 0.76a 8.46� 0.55c 6.40� 1.04c 6.01� 0.39c 36.41� 2.23c 55.771000 0.00c 3.10� 1.27 4.02� 0.88c 5.31� 0.38c 5.10� 1.15c 4.16� 0.34c 21.69� 3.49c 73.65ASA 100 0.00c 1.00� 0.00b 4.41� 0.90c 5.00� 0.12c 4.53� 1.28c 4.30� 0.45b 19.24� 2.75c 76.62
Data are expressed as mean� SEM. significant at ap50.05, bp50.01, cp50.001 when compared to control. n¼ 6.
Table 3. Effect of Homalium letestui stem extract on xylene-induced ear edema in mice.
Treatment/dose (mg/kg) Weight of right ear (g) Weight of left ear (g) Increase in ear weight (g) % Inhibition
Control (normal saline) 0.2 ml 0.074� 0.01 0.041� 0.00 (55.40) 0.033� 0.01Extract500 0.060� 0.01 0.040� 0.01 (33.33) 0.020� 0.01NS 39.39750 0.055� 0.01 0.040� 0.01 (27.27) 0.015� 0.01NS 54.541000 0.044� 0.01 0.039� 0.01 (11.36) 0.005� 0.01a 84.84Dexamethasone 4.0 0.043� 0.01 0.038� 0.01 (11.62) 0.005� 0.00a 84.84
Figures in parentheses indicate % increase in ear weight. Significant at ap50.01 when compared with control. n¼ 6.
1462 J. E. Okokon et al. Pharm Biol, 2013; 51(11): 1459–1466
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Discussion
H. letestui is used traditionally for the treatment of various
illnesses such as infections and inflammatory conditions.
In this study, the ethanol extract of the stem was evaluated for
anti-inflammatory and analgesic activities using various
experimental models.
In the carrageenan-induced edema, the extract (500–
1000 mg/kg) was observed to exert a significant effect (8.3–
70.0%) on edema caused by carrageenan. The prominent
effects of the extract at the early stage of inflammation (1–2 h)
indicate effect probably on histamine, serotonin and kinnins
that are involved in the early stage of carrageenan-induced
edema (Vane & Booting, 1987). The extract further reduction
of the later stage of the edema may be due to its ability to
inhibit prostaglandin, which is known to mediate the second
phase of carrageenan-induced inflammation (Vane &
Booting, 1987). However, acetyl salicylic acid (ASA)
(100 mg/kg), a prototype NSAID, is a cyclooxygenase
inhibitor whose mechanism of action involves inhibition of
prostaglandin, produced considerable inhibition of the paw
swelling induced by carrageenan injection.
The extract also inhibited egg albumin-induced edema
considerably (10.0–71.42%), demonstrating that it can inhibit
inflammation by blocking the release of histamine and 5-HT,
two mediators that are released by egg albumin (Nwafor et al.,
2007). However, ASA, a cyclooxygenase inhibitor, reduced
significantly edema produced by egg albumin.
The stem extract exerted significant inhibition (39.39–
84.84%) of ear edema caused by xylene at all doses. This
suggests the inhibition of phospholipase A2 which is involved
in the pathophysiology of inflammation due to xylene (Lin
et al., 1992). However, dexamethasone, a steroid anti-
inflammatory agent, produced significant reduction in the
mean right ear weight of positive control rats indicating an
inhibition of PLA2.
The extract significantly reduced acetic acid-induced
writhing, formalin-induced hind paw licking as well as
delayed the reaction time of animals (mice) to thermally
induced pain with inhibitory percentage ranges of 44.22–
73.65, 55.89–79.21 and 93.0–214.5%, respectively. Acetic
acid causes inflammatory pain by inducing capillary perme-
ability (Amico-Roxas et al., 1984; Nwafor et al., 2007) and in
part through local peritoneal receptors from peritoneal fluid
concentration of PGE2 and PGF2a (Bentley et al., 1983;
Deraedt et al., 1980). The acetic acid-induced abdominal
writhing is a visceral pain model in which the processor
releases arachidonic acid via cyclooxygenase, and prosta-
glandin biosynthesis plays a role in the nociceptive mechan-
ism (Franzotti et al., 2002). It is used to distinguish between
central and peripheral pain. These results suggest that the
extract may be exerting its action partly through the
lipoxygenase and/or cyclooxygenase system.
The organic acid has also been suggested to induce the
release of endogenous mediators indirectly, which stimulates
the nociceptive neurons that are sensitive to NSAIDs and
narcotics (Adzu et al., 2003). The inhibition of acetic acid-
induced writhing by the extract at all the doses suggests an
antinociceptive effect that might have resulted from the
inhibition of the synthesis of arachidonic acid metabolites.
Formalin-induced pain involves two different types of
pains which are in phases, neurogenic and inflammatory (Vaz
et al., 1996, 1997), and measures both centrally and periph-
erally mediated activities that are characteristic of biphasic
pain response. The first phase (0 to 5 min), named the
neurogenic phase, results from chemical stimulation that
provokes the release of bradykinin and substance P, while the
second and late phase initiated after 15 to 30 min of formalin
injection results in the release of inflammatory mediators
such as histamine and prostaglandins (Lu et al., 2008; Ridtitid
et al., 2008). The injection of formalin has been reported to
cause an immediate and intense increase in the spontaneous
activity of C-fiber afferent (pain-conducting nerve fiber) and
evokes a distinct quantifiable behavior indicative of pain
demonstrated in paw licking by the animals (Heapy et al.,
1987). The first phase of formalin-induced hind paw licking is
selective for centrally acting analgesics such as morphine
(Berken et al., 1991), while the late phase of formalin-induced
hind paw licking is peripherally mediated. Analgesic (noci-
ceptive) receptors mediate both the neurogenic and non-
neurogenic pain (Lembeck & Holzer, 1979). The extract
ability to inhibit both phases of formalin-induced paw licking
suggests its central and peripheral activities as well as its
ability to inhibit bradykinins, substance P, histamine and
prostaglandins, which are mediators in these pains.
Table 5. Effect of Homalium letestui stem extract on formalin-induced hind paw licking in mice.
Time intervals (min)
Treatment/dose (mg/kg) 5 10 15 20 25 30 Total % Reduction
Control 37.14� 0.11 15.25� 0.42 15.10� 0.44 14.34� 0.22 9.24� 0.14 7.28� 0.15 97.35� 1.48Extract500 22.15� 0.88 6.22� 0.45a 4.04� 0.48a 3.72� 0.27a 3.15� 0.25a 3.66� 0.21a 42.94� 2.54a 55.89750 24.16� 0.28a 3.76� 0.12a 2.94� 0.21a 3.15� 0.49a 2.14� 0.36a 3.00� 0.36a 39.15� 1.82a 59.781000 14.5� 2.74a 0.16� 0.23a 0.39� 0.14a 1.46� 0.12a 1.22� 0.51a 2.50� 0.22a 20.23� 3.96a 79.21ASA 100 8.83� 0.43a 1.03� 0.34a 1.83� 0.69a 1.12� 0.22a 1.02� 0.12a 0.09� 0.92a 13.92� 0.43a 85.70
Table 6. Effect of Homalium letestui stem extract on hot plate test.
GroupDose
mg/kg
Reactiontime (sec)
(mean� SEM) % Inhibition
Control – 3.86� 0.22H. letestui 500 7.45� 0.15a 93.00
750 9.28� 0.24a 140.411000 12.14� 0.42b 214.50
ASA 100 15.22� 0.12b 294.30
Data are expressed as mean� SEM. Significant at ap50.05, bp50.01when compared to control. n¼ 6.
DOI: 10.3109/13880209.2013.799707 Biological activities of Homalium letestui 1463
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The study also shows that the extract significantly delayed
the reaction time of the thermally induced (hot plate) test.
This model is selective for centrally acting analgesics and
indicates narcotic involvement (Turner, 1995) with opiod.
The GC–MS analysis has revealed the presence of vital
pharmacologically active compounds such as salicyl alcohol,
vanillin, 4-(3-hydroxy-1-propenyl)-2-methoxyphenol and
4-hydroxy-3, 5-dimethoxybenzaldehyde, a syringaldehyde,
that are potent anti-inflammatory and antinociceptive agents.
Salicyl alcohol (saligenin) belongs to the salicylates group,
which are known for anti-inflammatory and analgesic
activities due to inhibition of COX-1 and COX-2 (Rang
et al., 2007). Vanillin has been reported to inhibit
cyclooxygenase-2 (COX-2) (Murakami et al., 2007) and
possesses anti-inflammatory activity (Liang et al., 2009; Lim
et al., 2008; Murakami et al., 2007; Wu et al., 2009), as well
as antioxidant and free radical scavenging ability (Kamat
et al., 2000; Kumar et al., 2002; Lirdprapamongkol et al.,
2009) which could possibly account for its anti-inflamma-
tory action. However, its anti-inflammatory action is due to
its ability to inhibit inflammatory mediators (Lim et al.,
2008) and inhibit COX-2 because compounds with COX-2-
inhibiting activity possess anti-inflammatory properties
(Murakami et al., 2007).
Syringaldehydes present in H. letestui are also in Casearia
membranacea Hance (Flacourtiaceae) (Chang et al., 2003).
They are reported to exert inhibitory effects on cyclooxygen-
ase-2 (COX-2) (Deng et al., 2000) and prostaglandin synthase
(Stanikunaite et al., 2009) as well as ethyl phenylpropionate-
induced edema of the rat ear (Farah & Samuelsson, 1992).
Their antioxidant activity has equally been reported (Farah &
Samuelsson, 1992). Their presence in this extract may have
contributed to the observed anti-inflammatory and analgesic
activities.
The products of the COX and LOX pathways are involved
in the pathogenesis of several diseases, especially inflamma-
tory diseases. The LOX pathway produces leukotriene B4
(LTB4) that is the main leukotriene that plays a major role in
the inflammatory response (Hudson et al., 1993). 5-LOX is
the first and the key enzyme involved in the arachidonic acid
pathway to produce leukotrienes (Zhang et al., 2002). Some
plants, including Homalium panayanum, have been reported
to inhibit lipoxygenase (Chung et al., 2009). Because this
plant belongs to the same genus with H. letestui, there is a
possibility that H. letestui may also possess LOX-inhibiting
ability principles, thereby exerting effects observed in this
study. The combined activities of the chemical constituents of
this plant, especially vanillin and syringaldehyde, in inhibit-
ing inflammatory mediators, COX-2 and LOX enzymes
coupled to their antioxidant activities may have accounted
for the observed anti-inflammatory and analgesic activities.
Some terpenes, flavonoids and polyphenolic compounds
have also been revealed by GC–MS analysis to be present in
the plant extract. Flavonoids are known anti-inflammatory
compounds acting through inhibition of the cyclooxygenase
pathway (Liang et al., 1999). Some flavonoids are reported to
block both the cyclooxygenase and lipoxygenase pathways of
the arachidonate cascade at relatively high concentrations,
while at lower concentrations they only block lipoxygenase
pathway (Carlo et al., 1999). Some flavonoids exert their
antinociception via opioid receptor activation activity (Otuki
et al., 2005; Rajendran et al., 2000; Suh et al., 1996).
Flavonoids also exhibit inhibitory effects against phospholip-
ase A2 and phospholipase C (Middleton et al., 2000), and
cyclooxygenase and/or lipoxygenase pathways (Robak et al.,
1998).
Triterpenes have been implicated in anti-inflammatory
activity of plants (Huss et al., 2002; Suh et al., 1998) and
reports on their analgesic activities have also been published
(Krogh et al., 1999; Liu, 1995; Maia et al., 2006; Tapondjou
et al., 2003). Ursolic acid is a selective inhibitor of
cyclooxygenase-2 (Ringbom et al., 1998). Oleanolic acid is
known to exert its analgesic action through an opioid
mechanism, and possibly, a modulatory influence on vanilliod
receptors (Maia et al., 2006).
The extract has been reported above to exhibit anti-
inflammatory and analgesic activities. The presence of these
compounds (polyphenolics, flavonoids and triterpenes) in this
Table 7. GC–MS analysis of dichloromethane fraction of Homalium letestui.
S/No. Name of compound Mol. wt Chemical formula RI Concentration
1. 2,4 Heptadien-6-ynal,(E,E) 106 C7H6O 25 0.9122. 2-(4-Formyphenyloxy)-acetic acid 180 C9H8O4 172 0.2063. 2-Coumaranone 134 C8H6O2 195 0.9684. Benzoic acid 122 C7H6O2 200 0.2025. 4-Hepten-3-one,5-methyl,(E)- 126 C8H14O 207 8.8766. Salicyl alcohol 124 C7H8O2 234 3.2867. Vanillin 152 C8H8O3 312 0.9158. 3,4,5-Trimethoxy phenol 184 C9H12O4 456 5.7619. 2,4 Decadienal,(E,Z) 152 C10H16O 428 0.872
10. 4-(3-Hydroxy-1-propenyl)-2-methoxyphenol (E) 180 C10H12O3 527 2.01711. 4-Hydroxy-3,5-dimethoxybenzaldehyde 182 C9H10O4 477 0.86612. 5,6-Dimethoxyphthalaldehydic acid 210 C10H10OS 662 0.63413. 1-Methyl-4-(methylsulfinyl)benzene. 154 C8H10OS 596 0.59514. 4-Phenyl isocoumarin 222 C15H10O2 697 2.14715. 2,4-Dinitrophenylhydrazinebutanal 252 C10H12N4O4 743 0.28116. 1,3-Dihyroxy-4-methyl-9H-xanthen-9-one 242 C14H10O4 789 1.58017. (2,4-Dihydroxyphenyl)phenylmethanone 214 C13H10O3 805 2.14718. 1,2,3,4-Tetrahydro-5,8-dimethoxy-9,10-anthracenedione 272 C16H16O4 950 1.49119. Camphor 327 C19H21NO4 1153 5.58920. a-Terpineol 154 C10H18O 1185 15.642
1464 J. E. Okokon et al. Pharm Biol, 2013; 51(11): 1459–1466
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plant may have accounted for these activities and may in part
explain the mechanisms of its actions in this study.
In conclusion, the results of this study demonstrated that
H. letestui possesses anti-inflammatory and analgesic proper-
ties. Further investigation is being advocated especially in
elucidating cellular mechanisms and establishing structural
components of the active ingredients with a view of
standardizing them.
Acknowledgements
Dr. Jude Okokon is grateful to TWAS for financial support for
postdoctoral fellowship and ICCBS for providing research
facilities.
Declaration of interest
There is no conflict of interest.
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