Indian Journal of Experimental Biology Vol.49, September 2011, pp. 698-703

Analgesic activity of the aqueous seed extract of (K. Schum.) Hallier f. in rodents Olufunmilayo Olaide Adeyemi 1, *, Adejuwon Adewale Adeneye 1,2, * & Tope Elizabeth Alabi 1 1Department of Pharmacology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Idi-Araba, Lagos State, Nigeria 2Department of Pharmacology, Faculty of Basic Medical Sciences, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria

Received 6 April 2011; revised 24 June 2011

The analgesic effect and possible mechanism(s) of action of 50-200 mg/kg of the aqueous seed extract of H. umbellata (HU) were investigated in different experimental models of analgesia using the tail flick, tail immersion, acetic acid-induced writhing tests and formalin-induced algesia. Oral pre-treatment with 50-200 mg/kg of HU caused significant and dose related analgesic effect in the treated rats in all the experimental models used. This analgesia was mediated via central and peripheral mechanisms. Overall, the results showed that HU possesses analgesic effect which lends support to its folkloric use in the local management of pain.

Keywords: Analgesic activity, Aqueous seed extract, Hunteria umbellata , Rodents

Hunteria umbellata (K. Schum.) Hallier f. () Also, the analgesic and antipyretic effects of the is a tropical rainforest glabrous tree having smaller aqueous extract of the fruit pulp of H. umbellata flowers and fruits, and broad and elongated have been investigated and proven to be effective in leaves, measuring about 10-20 cm × 3.5-10 cm and the regulation of pain and fever and these effects ubiquitous to the West African and Central African were independent of its antibacterial activities 5. rainforests 1. Its fruit measures up to 5-25 cm in Hunteria umbellata have also been shown to be very diameter and consists of two separate globose mericaps active against micro-organisms such as Escherichia which are about 3-6 cm long, yellow, smooth, 8-25 coli , Proteus spp and Staphylococcus aureus 6. Oral seeded and embedded in a gelatinous pulp 2. hypoglycaemic activity of 50-200 mg/kg of the Different parts of the are employed by the aqueous seed extract of H. umbellata in various African traditional herbalists in the local models of hyperglycaemia, which was mediated via management of veterinary and human diseases. For inhibition of intestinal glucose uptake, inhibition of example, cold water decoction made from fresh adrenergic mechanism, increased peripheral glucose leaves of the plant is highly valued by traditional uptake and improvements in insulin resistance has midwives in the induction and augmentation of been recently reported 7,8 . labour while water decoction made from the seeds of In view of its folkloric use in pain management, its mature and ripe fruits is employed in the the present study has been designed at evaluating management of infections, stomach ulcers, diabetes, the analgesic property and the possible mechanism(s) and obesity 3. However, the folkloric use of the fresh of action of 50, 100 and 200 mg/kg of the aqueous leaves as an oxytocic agent was recently validated seed extract of H. umbellata in different experimental and its oxytocic action mediated via muscarinic models of analgesia. The chosen dose range acetycholinergic mechanism have also been (50-200 mg/kg) of the extract was made based on the established 3. The antinociceptive and antipyretic results of the preliminary study conducted earlier. effects of alkaloids extracted from the stem bark of have equally been reported 4. Materials and Methods Collection of plant materials—Fresh leaves, —————— *Correspondent authors inflorescence and mature fruits of the Hunteria Telephone (Mobile): +234-803-445-9618, +234-802-069-0946 umbellata (HU) plant were collected from the E-mail: [email protected]; [email protected] deciduous forest of Odorasanyin District of ADEYEMI et al .: ANALGESIC ACTIVITY OF HUNTERIA UMBELLATA AQUEOUS SEED EXTRACT 699

Ijebu-Igbo in Ijebu North Local Government Area time in seconds taken for each rat to flick its tail out of Ogun State, Nigeria, in June, 2009. Botanical of the water was noted as the reaction time. The rats identification and authentication were done as were screened before drugs were given and the only 7 previously described . those which flicked their tails before 60 s were used Plant extraction process—Hunteria umbellata in the experiment. Also the pre-latency time was seeds were harvested from the collected fresh ripe and taken and recorded. Measurements were taken for two mature fruits of the plant. The seeds were gently and a half hour at 30 min interval. washed in tap water and completely dried under room Tail immersion test—This test was based on the temperature (30º± 2ºC) for 4 weeks protected from modification of the method described by Janssen direct heat or sunlight. When dried, the seeds were et al 12 . Rats were randomly allotted to 5 groups of de-coated of their light-brown thin coatings. The dried 5 animals each. The lower portion of the tail was seed were then blended into fine powder using of immersed in a water bath maintained at 55º±0.5 oC. a domestic blender. Powdered sample (60 g) was Distilled water (10 mL/kg, po) was given to the dissolved in 1.5 L distilled water and left to stand in control group while the reference group was given the refrigerator for 72 h. After 72 h, the mixture was morphine (3 mg/kg, sc) the remaining groups were rigorously shaken but at intervals for 6 h and administered 50, 100 and 200 mg/kg, po of HU. The decanted. The solution was filtered using a piece of time in seconds for tail withdrawal from the water clean, white cloth and the filtrate was completely was taken as the reaction time, with a cut-off time air-dried in aerated oven preset at 40 oC to obtain of immersion set as 10 s. The reaction time was deep brown and sweet-smelling solid residue measured 30 min before and after administration of (yield was 40% w/w). The residue obtained was kept the drugs for two and a half hour. in air- and water-tight container and stored in the Acetic acid-induced mouse writhing test—This was 13 refrigerator and until required for experimentation. based on the method described by Koster et al . and 14 From the stock, a concentration of 100 mg/ml was Singh et al . Mice were randomly allotted to 5 groups prepared each time the extract is required for with 5 animals each. The control group received experiment. 10 mL/kg distilled water orally. The reference group received aspirin (100 mg/kg dissolved in distilled Experimental animals—Mature, white Albino water, po) and groups III, IV and V mice were Wistar rats weighing 100-150 g and Swiss albino orally pre-treated with 50, 100 and 200 mg/kg HU, mice weighing 16-20 g were obtained from the respectively. All drugs were administered 30 min Laboratory Animal House of the College of Medicine before ip injection of 0.6% (v/v), 1 mL/kg glacial of the University of Lagos, Nigeria, after the ethical acetic acid solution. The number of writhes (extension approval for the use of the animals have been of hind limb as a result of a wave of contraction of the obtained. The animals were allowed to acclimatize to abdominal muscles) immediately after injection of the laboratory conditions and given water and food ad acetic acid was counted for 30 min. A reduction in the libitum . All experimental animals were handled number of writhes is an indication of analgesic according to institutional and international guidelines property. guiding the use of experimental animals 9. Formalin test—The method adopted is as described Tests for analgesic activities of HU—Tail flick by Shibata et al 15 . Rats were divided into 6 groups method —The rat cold water tail flick test was based of 5 rats each. They were pre-treated with distilled 10 on a modification of the original method described water (Group I, 10 mL/kg, po); morphine (Group II, 11 by Pizziketti et al . Rats were randomly allotted to 10 mg/kg, ip) and 100 mg/kg of aspirin (Group III, 5 groups of 5 animals each. Pain was induced with 100 mg/kg, ip) and HU (Group IV, 50 mg/kg, po) o cold water at 0-1 C. Distilled water (10 mL/kg, po) (Group V, 100 mg/kg, po) (Group VI, 200 mg/kg, po). was given to the control group while the reference After 30 min, 0.03 mL of 1% formalin was injected group was given morphine (10 mg/kg, ip). The into the plantar aponeurosis of the right hind paw of remaining three groups were pre-treated orally with each rats. The licking or biting of the formalin 50, 100 and 200 mg/kg HU 30 min before subjecting injected right hind paw was counted in two phases. them to the tail flick test. Rats were restrained using The first phase was the first 5 min immediately after cylindrical wire gauze and the lower half of their tails the injection of the formalin and the second phase was immersed in a beaker of cold water (0º-1oC). The was 15-30 min after the injection of the formalin. 700 INDIAN J EXP BIOL, SEPTEMBER 2011

A reduction in the number of paw licking or biting is was recorded at 90 s compared with the untreated an indication of analgesic property. control. However, this effect was lower in all extract- Statistical analysis—Results are presented as the treated groups when compared with 10 mg/kg mean±SE. Data comparisons between treatment morphine-treated group (Table 2). groups were done by use of one-way ANOVA Acetic acid-induced mouse writhing test ─The followed by Dunnett’s post test. Values were number of writhes obtained in the control group considered statistically significant at P<0.05, <0.01, was 66.0 ± 1.8. Oral treatment with 50-200 mg/kg and <0.001. of HU caused a significant ( P<0.001) and dose- dependent inhibition of the acetic acid-induced Results writhes (Table 3). In addition, the inhibition (76.6%) Tail flick test—Oral pre-treatment with HU produced by 200 mg/kg the aqueous seed extract produced significant dose-dependent significant was significantly ( P<0.001) higher than that of (P<0.05) increase in reaction time to tail flick as time 100 mg/kg aspirin. progressed. The peak of the effect was recorded at Formalin test ─In the formalin-induced pain test, 120 s compared with the control. This effect was the extract caused a reduction in hind paw licking in significantly higher for 100 and 200 mg/kg of the both early and late phases of algesia. There was a extract compared with the control (Table 1). dose-dependent significant ( P<0.001) inhibition in Tail immersion test ─Similarly, oral treatment with phase two of the formalin-induced pain in extract 50-200 mg/kg of HU produced a significant dose- pre-treated rats. Percentage inhibition of the extract related ( P<0.05) prolongation of the reaction time to at 50 and 200 mg/kg were 59.5 and 56.0%, the tail immersion test. The peak effect of the extract respectively in the early phase of formalin test and

Table 1—Effect of 50-200 mg/kg of the aqueous seed extract of H. umbellata (HU) on rat tail flick test [Values are mean ± S.E. from 5 rats in each group. Figures in parentheses are % increase over control] Groups Reaction time (s) 0 30 60 90 120 150 I 28.8 ± 1.4 31.4 ± 1.0 33.4 ± 1.5 34.8 ± 1.3 36.8 ± 1.5 37.2 ± 1.8 II 27.6 ± 1.8 68.2 ± 6.6 c 76.4 ± 4.8 c 87.0 ± 1.6 c 89.0 ± 0.8 c 48.2 ± 4.6 (65.1) (78.2) (95.2) (98.4) (33.0) III 26.8 ± 1.9 42.8 ± 3.4 49.4 ± 3.5 54.8 ± 3.5 a 58.6 ± 3.3 b 44.6 ± 3.4 (25.3) (35.8) (44.3) (50.3) (21.2) IV 25.4 ± 1.0 50.8 ± 2.4 62.4 ± 6.0 b 69.4 ± 6.3c 78.4 ± 6.2 c 44.8 ± 7.0 (50.8) (62.4) (69.4) (78.4) (44.8) V 24.2 ± 1.0 65.8 ± 9.2 c 69.6 ± 8.5 c 75.6 ± 5.6 c 81.2 ± 3.8 c 45.0 ± 5.5 (63.2) (69.0) (78.1%) (86.6) (31.6%) a, b and c represent significant increases at P<0.05, <0.01, and <0.0001 respectively when compared to the control value. Group I= 10 mL/kg distilled water; Group II = 10 mg/kg morphine; Group III = 50 mg/kg HU; Group IV = 100 mg/kg HU; Group V = 200 mg/kg HU

Table 2—Effect of 50-200 mg/kg of the aqueous seed extract of H. umbellata (HU) on tail immersion test [Values are mean ± SE from 5 rats in each group. Figures in parentheses are % increase over control] Groups Reaction time (s) 0 30 60 90 120 150 I 108.0 ± 12.0 108.0 ± 12.0 84.0 ± 12.0 84.0 ± 12.0 84.0 ± 12.0 72.0 ± 12.0 II 108 ± 12.0 300.0 ± 30.0 c 300.0 ± 30.0 c 300.0 ± 18.0 c 156.0 ± 12.0 c 144.0 ± 12.0 (41.9) (41.9) (41.9) (14.0) (11.6) III 120.0 ± 0.0 144.0 ± 12.0 156.0 ± 12.0 a 156.0 ± 12.0 a 120.0 ± 0.0 b 96.0 ± 12.0 (5.0) (7.5) (7.5) (0.0) (-5.0) IV 120.0 ± 0.0 192.0 ± 30.0 a 204.0 ± 12.0 c 204.0 ± 12.0 c 132.0 ± 12.0 a 120.0 ± 0.0 a (15.0) (17.5) (17.5) (2.5) (0.0) V 97.2 ± 12.0 204.0 ± 12.0 a 216.0 ± 12.0 c 216.0 ± 12.0 c 144 ± 12.0 a 132.0 ± 12.0 b (21.4) (23.8) (23.8) (9.5) (7.1) a, b and c represent significant increases at P<0.05, <0.01, and <0.0001 respectively when compared to the control value. Group I= 10 mL/kg distilled water; Group II = 10 mg/kg morphine; Group III = 50 mg/kg HU; Group IV = 100 mg/kg HU; Group V = 200 mg/kg HU ADEYEMI et al .: ANALGESIC ACTIVITY OF HUNTERIA UMBELLATA AQUEOUS SEED EXTRACT 701

these inhibitions were higher than that of 100 mg/kg Discussion aspirin (Fig. 1). The extract also produced a dose Pain is an unpleasant sensory and emotional dependent reduction in hind paw licking in the experience associated with actual or potential tissue phase two of the formalin-induced pain. damage, or described in terms of such damage 16 . Pain is defined as a subjective, unpleasant, physical and Table 3—Effect of 50-200 mg/kg of the aqueous seed extract of psychological experience observed as a result of the H. umbellata on acetic acid-induced mouse writhing test stimulation of identifiable nerve fibres with defined [Values are expressed as mean ± SE of 5 rats in each group] pathways to the brain via the spinal cord 17 . Pain often Groups Treatment No. of writhing Inhibition of results from tissue damage that stimulates nociceptive per 30 min writhing (%) receptors (nociceptive pain) but pain may also I 10 mL/kg distilled water 66.0 ± 1.8 - occur without nociception, here it could be as a II 100 mg/kg aspirin 29.0 ± 0.9* 56.5 result of damage to neural structures (neuropathic pain or neuralgia). While the former is often acute, III 50 mg/kg HU 25.0 ± 0.8* 62.5 self- limiting after healing and responds easily to IV 100 mg/kg HU 20.4 ± 0.4* 69.4 analgesics, the latter is very difficult to treat, there V 200 mg/kg HU 15.6 ± 0.5* 76.6 may or may not be evidence of injury, causes chronic *represents a significant decrease at P<0.001 when compared to pain and will persist long after the initial injury has the control value healed 18,19 . The present study was aimed at investigating the anti-nociceptive property of the aqueous seed extract of H. umbellata , using the tail flick, tail immersion and the acetic acid-induced mouse writhing tests while the formalin-induced pain method was adopted so as to establish its mechanism(s) of action. In the tail immersion and tail flick tests, oral pre-treatment with the extract caused a profound and dose related analgesia in the treated rats although the analgesic effect of the extract was less effective than morphine. The above two procedures consists of behavioural methods that have been developed to study nociception in animals 20 . The animal response in these tests is usually integrated at the lower levels in the central nervous system, thus, giving information about the pain threshold. They are, therefore, used to detect narcotic and non-narcotic analgesics. It is well established that thermal nociceptive tests are more sensitive to opioid µ-agonists and non-thermal tests to opioid κ-agonists 21 . The data generated in the present study suggest that the involvement of both κ and µ opioid receptor in the analgesic activity of HU, from which the central involvement of the extract could be deduced. The formalin-induced pain as an experimental model of analgesia is useful for elucidating mechanism of pain and analgesia since it measures the response to a long-lasting nociceptive stimulus and, therefore, resembles clinical pain 22 . Subcutaneous injection of dilute formalin into mice Fig. 1—Effect of 50-200 mg/kg of the aqueous seed extract of hind-paw produces biphasic nociceptive response H. umbellata (HU) on the phases I and II of formalin-induced algesia in treated rats ( f represents a significant decrease in the namely: the first transient phase is caused by the number of paw lickings of treated rats when compared to the direct effect of formalin on sensory C-fibers, and untreated control values) the second prolonged phase is associated with 702 INDIAN J EXP BIOL, SEPTEMBER 2011

the development of the injury induced spinal analgesic mechanisms have been postulated. Further sensitization, responsible for facilitated pain studies, especially at the molecular level may provide processing, a central sensitization of the dorsal horn better understanding of its exact mode and site(s) neuron occurs during inflammatory pain 23-25 . Drugs of actions. In addition, isolation and characterization that act centrally, such as the narcotics inhibit both of the suspected analgesic phytocomponents will be phases of formalin-induced pain, while peripherally worthwhile in the nearest future. acting drugs such as aspirin only inhibit the late phase 25-27 . Results of the present study showed that References HU inhibit both the early and late phases of formalin 1 Boone M J Hunteria umbellata (K. 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