anti-inflammatory and antinociceptive activities of homalium letestui

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: [email protected]

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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.


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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.


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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


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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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anti-inflammatory and antinociceptive activities of homalium letestui

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