Anti-inflammatory and antinociceptive activities of Homalium letestui

Abstract

Context. Homalium letestui Pellegr (Flacourtiaceae) is used in various decoctions traditionally by the Ibibios of the Niger Delta of Nigeria to treat stomach ulcer, malaria and other inflammatory diseases, as well as an aphrodisiac.

Objective: To investigate the anti-inflammatory and antinociceptive activities of the stem extract of the plant.

Materials and methods: The ethanol stem extract (500, 750, 1000 mg/kg, i.p.) of H. letestui was investigated for anti-inflammatory activity using carrageenan, egg albumin-induced and xylene-induced ear edema models and analgesic activity using acetic acid-induced writhing, formalin-induced paw licking and thermal-induced pain models. The ethanol extract was administered to the animals orally, 30 min to 1 h depending on the model, before induction of inflammation/pain. The LD₅₀ was also determined. GC–MS analysis of dichloromethane fraction was carried out.

Results: The extract caused a significant (p < 0.05–0.001) reduction of inflammation induced by carrageenan (8.3–70.0%), egg albumin (10.0–71.42%) and xylene (39.39–84.84%). The extract also reduced significantly (p < 0.05–0.001) 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 of H. letestui possesses anti-inflammatory and analgesic properties which may in part be mediated through the chemical constituents of the plant as revealed by the GC–MS.

• Analgesic
• anti-angiogenic
• Flacourtiaceae
• phytochemicals

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 inflammatory 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 anthelmintic 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 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, dichloromethane, 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 committee, 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 manifestation of physical signs of toxicity such as writhing, decreased motor activity, decreased body/limb tone, decreased respiration 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 circumference between the control and 0.5, 1, 2, 3, 4 and 5 h after administration of phlogistic agent was used to assess inflammation (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 % = 100 × (Vc – Vt)/Vc, where Vc and Vt represent 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 % = 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 contraction 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 μl of 2.5% formalin solution (0.9% formaldehyde) made up in phosphate buffer solution (PBS concentration: NaCl 137 mM, KCl 2.7 mM and phosphate 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 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., 0.25 µm 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 1.0 µl, 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 (p < 0.05–0.001) anti-inflammatory effect which was comparable to the standard drug, ASA (100 mg/kg). The percentage reduction of inflammation was 8.3–70.0% (Table 1).

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 ± 0.01
Extract
500	0.22 ± 0.01	0.33 ± 0.01 (8.3)	0.35 ± 0.01^a (0.0)	0.32 ± 0.01^b (16.6)	0.30 ± 0.01^b (27.2)	0.28 ± 0.01^b (60.0)	0.27 ± 0.01^b (16.6)
750	0.24 ± 0.01	0.34 ± 0.01 (16.6)	0.34 ± 0.01^a (23.0)	0.31 ± 0.01^b (41.6)	0.29 ± 0.01^b (54.5)	0.27 ± 0.01^b (70.0)	0.26 ± 0.01^b (66.6)
1000	0.23 ± 0.01	0.34 ± 0.01 (8.3)	0.33 ± 0.01^b (23.0)	0.30 ± 0.01^b (41.7)	0.28 ± 0.01^b (54.5)	0.26 ± 0.01^b (70.0)	0.25 ± 0.01^b (66.6)
ASA 100	0.24 ± 0.01	0.34 ± 0.01 (16.6)	0.34 ± 0.01^a (23.0)	0.32 ± 0.01^b (33.3)	0.30 ± 0.01^b (45.5)	0.28 ± 0.01^b (60.0)	0.25 ± 0.01^b (83.3)

Data are expressed as mean ± SEM. Significant at ^ap < 0.05, ^bp < 0.001 when compared to control. n = 6. Values in parentheses represent % inhibition of inflammation.

Egg albumin-induced edema

Administration of stem extract of H. letestui (500–1000 mg/kg) caused a significant (p < 0.05–0.001) anti-inflammatory effect against edema caused by egg albumin in mice with a considerable % reduction of inflammation (10.0–71.42%). The effect was comparable to that of standard drug, ASA (100 mg/kg) (Table 2).

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 ± 0.01
Extract
500	0.25 ± 0.01	0.34 ± 0.01	0.34 ± 0.01 (10.0)	0.33 ± 0.01 (20.2)	0.31 ± 0.01^a (22.2)	0.29 ± 0.01^a (50.0)	0.28 ± 0.01^a (57.4)
750	0.25 ± 0.01	0.33 ± 0.01	0.33 ± 0.01 (20.0)	0.32 ± 0.01^a (30.0)	0.29 ± 0.01^a (55.5)	0.29 ± 0.01^b (50.0)	0.27 ± 0.01^b (71.4)
1000	0.23 ± 0.01	0.33 ± 0.01	0.33 ± 0.01 (0.0)	0.31 ± 0.01^a (20.0)	0.28 ± 0.01^b (44.4)	0.27 ± 0.01^b (50.0)	0.25 ± 0.01^b (71.4)
ASA 100	0.24 ± 0.01	0.35 ± 0.01^a	0.34 ± 0.01^a (0.0)	0.32 ± 0.01^b (20.0)	0.26 ± 0.01^b (77.7)	0.26 ± 0.01^b (75.0)	0.25 ± 0.01^b (85.7)

Data are expressed as mean ± SEM. Significant at ^ap < 0.01, ^bp < 0.001 when compared to control. n = 6. Values in parentheses represent % inhibition of inflammation.

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 (p < 0.01) 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).

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 ± 0.01
Extract
500	0.060 ± 0.01	0.040 ± 0.01	(33.33) 0.020 ± 0.01^NS	39.39
750	0.055 ± 0.01	0.040 ± 0.01	(27.27) 0.015 ± 0.01^NS	54.54
1000	0.044 ± 0.01	0.039 ± 0.01	(11.36) 0.005 ± 0.01^a	84.84
Dexamethasone 4.0	0.043 ± 0.01	0.038 ± 0.01	(11.62) 0.005 ± 0.00^a	84.84

Figures in parentheses indicate % increase in ear weight. Significant at ^ap < 0.01 when compared with control. n = 6.

Effect of ethanol crude extract of stem of H. letestui on acetic 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 (p < 0.001) relative to control and comparable to that of the standard drug, ASA, at the highest dose, 1000 mg/kg (Table 4).

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 ± 6.74
Extract	0.00^c	8.26 ± 0.73	10.01 ± 1.18^a	9.30 ± 1.06^c	10.11 ± 1.20^c	8.23 ± 0.72^c	45.91 ± 4.09^c	44.22
500
750	0.00^c	6.44 ± 0.80	9.10 ± 0.76^a	8.46 ± 0.55^c	6.40 ± 1.04^c	6.01 ± 0.39^c	36.41 ± 2.23^c	55.77
1000	0.00^c	3.10 ± 1.27	4.02 ± 0.88^c	5.31 ± 0.38^c	5.10 ± 1.15^c	4.16 ± 0.34^c	21.69 ± 3.49^c	73.65
ASA 100	0.00^c	1.00 ± 0.00^b	4.41 ± 0.90^c	5.00 ± 0.12^c	4.53 ± 1.28^c	4.30 ± 0.45^b	19.24 ± 2.75^c	76.62

Data are expressed as mean ± SEM. significant at ^ap < 0.05, ^bp < 0.01, ^cp < 0.001 when compared to control. n = 6.

Effect of ethanol stem extract of H. letestui on formalin-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 (p < 0.001) and comparable to that of the standard drug, ASA, at the highest dose, 1000 mg/kg (Table 5).

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 ± 1.48
Extract
500	22.15 ± 0.88	6.22 ± 0.45^a	4.04 ± 0.48^a	3.72 ± 0.27^a	3.15 ± 0.25^a	3.66 ± 0.21^a	42.94 ± 2.54^a	55.89
750	24.16 ± 0.28^a	3.76 ± 0.12^a	2.94 ± 0.21^a	3.15 ± 0.49^a	2.14 ± 0.36^a	3.00 ± 0.36^a	39.15 ± 1.82^a	59.78
1000	14.5 ± 2.74^a	0.16 ± 0.23^a	0.39 ± 0.14^a	1.46 ± 0.12^a	1.22 ± 0.51^a	2.50 ± 0.22^a	20.23 ± 3.96^a	79.21
ASA 100	8.83 ± 0.43^a	1.03 ± 0.34^a	1.83 ± 0.69^a	1.12 ± 0.22^a	1.02 ± 0.12^a	0.09 ± 0.92^a	13.92 ± 0.43^a	85.70

Data are expressed as mean ± SEM. Significant at ^ap < 0.001. when compared to control. n = 6.

Effect of ethanol crude extract of stem of H. letestui on thermally induced pain in mice

The stem extract (500–1000 mg/kg) exhibited a considerable effect on thermally induced pain in mice. This inhibition was statistically significant (p < 0.001) relative to the control (Table 6). The percentage inhibition range was 93.0–214.5%.

Table 6. Effect of Homalium letestui stem extract on hot plate test.

Group	Dose mg/kg	Reaction time (sec) (mean ± SEM)	% Inhibition
Control	–	3.86 ± 0.22
H. letestui	500	7.45 ± 0.15^a	93.00
	750	9.28 ± 0.24^a	140.41
	1000	12.14 ± 0.42^b	214.50
ASA	100	15.22 ± 0.12^b	294.30

Data are expressed as mean ± SEM. Significant at ^ap < 0.05, ^bp < 0.01 when compared to control. n = 6.

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 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	0.912
2.	2-(4-Formyphenyloxy)-acetic acid	180	C9H8O4	172	0.206
3.	2-Coumaranone	134	C8H6O2	195	0.968
4.	Benzoic acid	122	C7H6O2	200	0.202
5.	4-Hepten-3-one,5-methyl,(E)-	126	C8H14O	207	8.876
6.	Salicyl alcohol	124	C7H8O2	234	3.286
7.	Vanillin	152	C8H8O3	312	0.915
8.	3,4,5-Trimethoxy phenol	184	C9H12O4	456	5.761
9.	2,4 Decadienal,(E,Z)	152	C10H16O	428	0.872
10.	4-(3-Hydroxy-1-propenyl)-2-methoxyphenol (E)	180	C10H12O3	527	2.017
11.	4-Hydroxy-3,5-dimethoxybenzaldehyde	182	C9H10O4	477	0.866
12.	5,6-Dimethoxyphthalaldehydic acid	210	C10H10OS	662	0.634
13.	1-Methyl-4-(methylsulfinyl)benzene.	154	C8H10OS	596	0.595
14.	4-Phenyl isocoumarin	222	C15H10O2	697	2.147
15.	2,4-Dinitrophenylhydrazinebutanal	252	C10H12N4O4	743	0.281
16.	1,3-Dihyroxy-4-methyl-9H-xanthen-9-one	242	C14H10O4	789	1.580
17.	(2,4-Dihydroxyphenyl)phenylmethanone	214	C13H10O3	805	2.147
18.	1,2,3,4-Tetrahydro-5,8-dimethoxy-9,10-anthracenedione	272	C16H16O4	950	1.491
19.	Camphor	327	C19H21NO4	1153	5.589
20.	α-Terpineol	154	C10H18O	1185	15.642

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 permeability (Amico-Roxas et al., 1984; Nwafor et al., 2007) and in part through local peritoneal receptors from peritoneal fluid concentration of PGE₂ and PGF_2α (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 prostaglandin biosynthesis plays a role in the nociceptive mechanism (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 peripherally 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 (nociceptive) 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.

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-inflammatory 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 cyclooxygenase-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 inflammatory 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 inhibiting 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 phospholipase 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 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 properties. Further investigation is being advocated especially in elucidating cellular mechanisms and establishing structural components of the active ingredients with a view of standardizing them.

Declaration of interest

There is no conflict of interest.

Acknowledgements

Dr. Jude Okokon is grateful to TWAS for financial support for postdoctoral fellowship and ICCBS for providing research facilities.