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Evaluation of the antinociceptive activity of Ficus deltoidea aqueous extract
Fitoterapia 79 (2008) 557 – 561 www.elsevier.com/locate/fitote
Evaluation of the antinociceptive activity of Ficus deltoidea aqueous extract M.R. Sulaiman a,⁎, M.K. Hussain a , Z.A. Zakaria b , M.N. Somchit a , S. Moin a , A.S. Mohamad a , D.A. Israf a a
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b Department of Pharmacology, Faculty of Pharmacy, Universiti Teknologi Mara, 40450 Shah Alam, Selangor, Malaysia Received 11 June 2008; accepted 18 June 2008 Available online 10 July 2008
Abstract The aqueous extract of Ficus deltoidea leaves was evaluated for possible antinociceptive activity in three models of nociception, namely, acetic acid-induced abdominal writhing, formalin and hot plate test. The results of the present study showed that intraperitoneal administration of the F. deltoidea leaves aqueous extract at the dose of 1, 50 and 100 mg/kg, 30 min prior to pain induction produced significant dose-dependent antinociceptive effect in all the models used, which indicating the presence of both central and peripherally mediated activities. Furthermore, the antinociceptive effect of the extract in the formalin and hot plate test was reversed by the non-selective opioid receptor antagonist naloxone suggesting that the endogenous opioid system is involved in its analgesic mechanism of action. Thus, the present results demonstrated that F. deltoidea leaves aqueous extract contains pharmacologically active constituents which possess antinociceptive activity justifying its popular therapeutic use in treating conditions associated with the painful conditions. © 2008 Elsevier B.V. All rights reserved. Keywords: Ficus deltoidea; Antinociceptive; Acetic acid-induced abdominal constriction test; Formalin test; Hot plate test
1. Introduction Ficus deltoidea is an epiphytic shrub which is native and widely distributed in several countries of the Southeast Asia. In Malaysia, it is commonly known as Mas cotek, serapat angin, telinga beruk and other names [1]. Different parts of the plant are used traditionally to treat various kinds of ailments.The fruits are chewed to relieve headache, toothache and cold; powdered root and leaves of the plant has been applied externally to wounds and sores, and around the joints for relief of rheumatism. Decoction or infusion from the whole plants is a well known traditional herbal drink for women after childbirth to help strengthen the uterus. Moreover it improves blood circulation, regain energy and it is believe to enhance sexual desire (personal communication from local people where the plant was collected). Besides being one of the popular herbs used in Malay traditional medicine, the pharmacological properties of this plant have not yet been studied, only ⁎ Corresponding author. Tel.: +6 03 89472603; fax: +6 03 89472585. E-mail address: [email protected] (M.R. Sulaiman). 0367-326X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.06.005
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one report of blood glucose lowering effect has been published [2]. The basis for the traditional use of this herb in the management of painful related conditions has not yet been scientifically verified to the best of our knowledge. The present study therefore, is aimed at investigating possible antinociceptive activity of the aqueous extract from the leaves of F. deltoidea and to explore its possible mechanism of action using chemical and thermal nociception models. 2. Experimental 2.1. Plant material The F. deltoideae fresh whole plant (Moraceae), collected from its natural habitat in Sepang, Selangor, Malaysia was identified by Mr. Shamsul Khamis. A voucher specimen (SK1510/2007) was deposited in the Phytomedicinal Herbarium, Institute of Biosciences, Universiti Putra Malaysia, Serdang, Selangor. 2.2. Aqueous extract The F. deltoideae leaves aqueous extract (FDA) was prepared as previously described [3]. Briefly, powdered dried leaves (100 g) infused in distilled water 60 °C for 2 days, filtered, and lyophilized yielding a dark brown colored semisolid mass (yields17.5%). The extract was kept at − 20 °C. 2.3. Animals Experiments were conducted using adult male Balb C mice (25–35 g) and adult male Sprague–Dawley rats (200–250 g), housed at 22± 2 °C under a 12-h light/12-h dark cycle and with access to food and water ad libitum. The animals were acclimatized to the laboratory for at least 1 h before testing and were used only once throughout the experiments. Experiments reported in this study were performed in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals [4]. 2.4. Preliminary phytochemical screening Phytochemical screening of the FDA was performed to detect the presence of different classes of constituents, such as alkaloids, flavonoids, saponins, steroids, terpenes and tannins [5,6]. 2.5. Antinociceptive tests 2.5.1. Acetic acid-induced abdominal writhing test The acetic acid-induced abdominal writhing test was performed according to procedures described previously [3,7]. Mice received the FDA (1, 50, and 100 mg/kg, i.p.) or normal saline 30 min prior to injection of 0.6% acetic acid (10 ml/kg, i.p.). The number of writhing was recorded for 30 min, starting 5 min after the injection of acetic acid. Acetylsalicylic acid (ASA, 100 mg/kg, i.p.) was administrated 30 min before the nociceptive agent. 2.5.2. Formalin test The test was performed according to Dubuisson and Dennis [8]. Briefly, 30 min after injections of the FDA (1, 50, and 100 mg/kg, i.p.) and ASA (100 mg/kg, i.p.) or normal saline, 50 μl of formaldehyde 2.5% (v/v in distilled water) was injected subcutaneously into the plantar surface of the left hind paw of the rats. The behavioral responses to nociception including biting, licking and scratching of the injected paw were noted and the time spent was recorded up to 30 min. The first 5 min was considered as early phase (neurogenic phase) and the period of 15–30 min as the late phase (inflammatory phase) of the nociceptive response. 2.5.3. Hot plate test The hot plate test was used to measure the response latencies according to the method described previously [3]. In these experiments, the hot plate (Ugo Basile, model-7280) was maintained at 55 ± 2 °C. Animals were placed into the perspex cylinder on the heated surface, and the time between placement and licking of the hind paws or jumping
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movements was recorded as response latency. The FDA (1, 50, 100 mg/kg, i.p.) and morphine (5 mg/kg, i.p.) were administered 30 min before the beginning of the experiment.Mice were observed before and at 30, 60, 120, 180, 240 and 300 min after substance administration. The cut-off time was 20 s. 2.5.4. Involvement of opioid receptors In order to verify the involvement of opioidergic system in the FDA-induced antinociception, separated groups of mice and rats (n = 6) were pre-treated with non-selective opioid receptor antagonist, naloxone (5 mg/kg, i.p), which was injected 15 min before the administration of the extract (100 mg/kg; i.p.) and morphine (5 mg/kg), and tested using the hot plate and the formalin testas described above. 2.6. Statistical analysis The statistical analyses were performed by ANOVA, followed by Dunnett's multiple comparison tests. The results were expressed as the mean ± S.E.M. to show variation in groups. Differences are considered significant when P ≤ 0.05. 3. Results and discussion The present study was conducted to assess the antinociceptive effectof the F. deltoidea, a medicinal claimed in Malay folk medicine for its analgesic property. It was demonstrated on chemical nociception in the test models of acetic acid-induced writhing, formalin-induced paw licking and on thermal nociception in hot plate test. These methods were selected such that both centrally and peripherally mediated effects were investigated. The acetic acid-induced abdominal constriction is believed to show the involvement of peripheral mechanisms, whereas the hot plate test is believed to show that of central mechanisms [9]. The formalin test is used to investigate both peripheral and central mechanisms [10]. Results of the present study demonstrated that the FDA possessed antinociceptive activity evident in all the nociceptive models, which suggestive of the presence of both central and peripherally mediated activities. In the acetic acid-induced abdominal constriction test, the results showed that the FDA extract (1, 50 and 100 mg/kg) potently and significantly reduced the number of abdominal writhing in a dose dependent manner with 50.6%, 70.4% and 88.0% of inhibition, respectively as compared to control animals (Table 1). The positive control group treated with acetylsalicylic acid (100 mg/kg) also manifested significantly reduction on the number of writhes (61.5%). It has been postulated that acetic acid acts indirectly by inducing the release of endogenous mediators, such as PGE2 (prostaglandin E2) and PGF2α in peritoneal fluids as well as lipooxygenase products, which stimulate the nociceptive neurons sensitive to NSAIDs [11,12]. Therefore, the result of the acetic acid-induced writhing strongly suggests that the mechanism of this extract may be linked partly to inhibition of lipooxygenase and/or cyclooxygenase in peripheral tissues, thereby reducing PGE2 synthesis and interfering with the mechanism of transduction in primary afferent nociceptor. In the formalin test which is sensitive for various classes of analgesic drugs [13], administration of the extract demonstrated significant dose dependent inhibition in both early (27.7%, 36.5%, and 68.5%) and late phase (31.9%, 44.5%, 72.5%) licking responses, at the tested doses of 1, 50 and 100 mg/kg, respectively (Table 2). Similarly, morphine produced marked inhibition of both the early phase (90.4%) and late phase (88.5%) of the formalin test. In contrast, the treatment of animals with ASA caused significant inhibition (73.3%) of the late phase, but not the early phase. It is also well known that the formalin test may involve sensorial C-fibers [14] in early phase and a combined Table 1 Effect of the F. deltoideae aqueous extract (FDA) on acetic acid-induced abdominal writhing test in mice Material Control (NaCl, 10 ml/kg, i.p.) FDA
Acetylsalicylic acid
Dose (mg/kg; i.p.)
No. of writhings
Inhibition (%)
1.0 50.0 100.0 100.0
62.0 ± 3.9 30.6 ± 2.7⁎ 18.4 ± 3.4⁎⁎ 7.4 ± 2.4⁎⁎ 24.0 ± 3.5⁎
– 50.6 70.4 88.0 61.5
Values are mean ± S.E.M.; (N = 10). ⁎ P b 0.05; ⁎⁎P b 0.01 significantly different from control (ANOVA followed by Dunnett's test).
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Table 2 Effect of the F. deltoideae aqueous extract (FDA) on formalin-induced pain in rats Material
Dose (mg/kg; i.p.)
Control (NaCl, 10 ml/kg, i.p.) FDA
Acetylsalicylic acid Morphine Morphine + Naloxone FDA + Naloxone
Total time spent in licking (s)
1.0 50.0 100.0 100.0 5 5+5 50 + 5
0–5 min
Inhibition (%)
15–30 min
Inhibition (%)
57.2 ± 11.0 41.3 ± 2.3⁎ 36.3 ± 2.3⁎ 18.0 ± 2.6⁎⁎ 51.8 ± 7.2 5.5 ± 0.6⁎⁎ 52.2 ± 2.3⁎# 50.6 ± 7.7⁎#
– 27.7 36.5 68.5 9.4 90.4 8.7 11.5
140.9 ± 8.4 96.0 ± 3.7⁎ 78.2 ± 4.6⁎ 38.7 ± 5.3⁎⁎ 37.6 ± 8.8⁎⁎ 16.2 ± 1.2⁎⁎ 129.6 ± 3.1⁎# 109.5 ± 9.7⁎#
– 31.9 44.5 72.5 73.3 88.5 8.0 22.3
Results are expressed as the mean ± S.E.M. in seconds (N = 10). ⁎P b 0.05; ⁎⁎P b 0.01 compared to the control group. # P b 0.05 compared to the group receiving appropriate drug/extract at the same dose without naloxone. (Dunnet's test).
process generated by peripheral inflammatory tissue and functional changes in the dorsal horn in late phase [15,16]. The centrally acting drugs such as narcotics inhibited both phases equally, while peripherally acting drugs only inhibited the second phase [17]. Inhibition of both phases of pain as observed with the extract in this study showed that they contain active analgesic principles acting both centrally and peripherally. The central analgesic effect of the aqueous extract may be supported by the results recorded in the hot plate test which is a selective method able to screen centrally acting opiate analgesic drugs [9]. It was demonstrated that intraperitoneal administration of the extract (1, 50 and 100 mg/kg) exerts significant prolonged in the response latency time to the heat stimulus (Table 3). This effect begun early at 30 min after administration of aqueous extract and persists until the following fifth hour. As expected, morphine (5 mg/kg, i.p.) significantly increased the latency time to the nociceptive response compared with control group. It is also interesting to note that, pre-treatment with a non-selective opioid receptor antagonist, naloxone significantly reversed the antinociceptive effect of FDA extract and morphine in both phases of the formalin test as well as in the hot plate test, compared to extract and morphine alone (Tables 2 and 3). These results suggest that activation of central opioid receptors are involved in the antinociceptive action of the FDA extract. Preliminary phytochemical screening carried out on this species showed the presence of numerous constituents' classes, such as flavonoids, saponins, tannins, steroids and terpenes. Since several flavonoids and tannins isolated from medicinal plants have been discovered for their significant antinociceptive and/or anti-inflammatory activity [18–20]. It is, therefore, possible that the antinociceptive effects observed with this extract in the present study may be attributable to its flavonoids and tannins component. In addition, the preliminary acute oral toxicity test obtained with this plant showed no death occurred, even at the highest dose of FDA (5 g/kg), indicating it may have a reasonable safety margin with regards to acute toxicity (data not shown). This further justifying its wide application in treatment of pain in various disease conditions and lack of any reported side effects with the traditional use of this plant. Table 3 Effect of the F. deltoideae aqueous extract (FDA) on the hot plate test in mice Material
Control (NaCl) FDA
FDA + Naloxone Morphine Morphine + Naloxone
Dose (mg/kg)
1 50 100 100 + 5 5 5+5
Latency time 0
30
60
120
180
240
300
6.4 ± 1.2 7.2 ± 0.5 6.5 ± 0.3 6.9 ± 1.5 6.6 ± 0.3 7.9 ± 1.6 6.5 ± 1.1
7.3 ± 0.6 8.5 ± 1.1⁎ 9.4 ± 0.3⁎ 11.9 ± 0.6⁎ 7.54 ± 0.3# 17.1 ± 0.3⁎ 8.4 ± 2.2#
7.8 ± 0.6 9.4 ± 0.2⁎ 11.0 ± 0.6⁎ 11.9 ± 1.6⁎ 6.89 ± 0.3# 18.1 ± 0.6⁎ 8.3 ± 1.3#
7.5 ± 1.1 9.6 ± 0.3⁎ 12.7 ± 0.5⁎ 14.8 ± 0.5⁎ 7.17 ± 0.7# 19.0 ± 2.2⁎ 8.0 ± 1.5#
6.9 ± 1.6 8.4 ± 0.6⁎ 11.5 ± 1.2⁎ 13.9 ± 0.3⁎ 7.53 ± 2.3# 16.8 ± 0.3⁎ 8.5 ± 0.3#
7.4 ± 0.5 9.5 ± 0.3⁎ 12.9 ± 0.6⁎ 14.7 ± 1.3⁎ 7.67 ± 0.6# 15.4 ± 0.4⁎ 10.3 ± 1.6#
6.5 ± 0.3 7.3 ± 0.3 10.0 ± 1.3⁎ 11.2 ± 0.3⁎ 6.24 ± 1.3# 14.2 ± 0.3⁎ 7.5 ± 0.3#
Results are expressed as the mean ± S.E.M. in seconds (N = 8). ⁎P b 0.05 compared to the control group. # P b 0.05 compared to the group receiving appropriate drug/extract at the same dose without naloxone (Dunnet’s test).
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In conclusion, the present study demonstrated that the aquoues extract of FD possess significant antinociceptive effects in laboratory animals at the doses investigated. The results seems to support the traditional use of this plant in some painful conditions and also suggest the presence of biologically active principles which may be worth further investigation and elucidation. Further studies are in fact currently under way to isolate and characterize the active principle(s) of the crude extract. Acknowledgement The authors would like to thank the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for providing the necessary support for the study. This research was supported by a Research University Grant Scheme 2007 (04/01/07/0093RU) from the Universiti Putra Malaysia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
Musa Y. Bul Teknol Tanaman 2005;2:35. Aminudin N, Sin CY, Chee ES, Nee KI, Renxin L. Malays J Sci 2007;26:73. Sulaiman MR, Somchit MN, Israf DA, Ahmad Z, Moin S. Fitoterapia 2004;75:667. Zimmermann M. Pain 1983;16:109. Wagner H, Bladt S, Zgainski EM. Plant drug analysis. Berlin: Springer; 1984. Trease GE, Evans WC. Pharmacognosy. 12th ed. London: ELBS Publication; 1985. Koster R, Anderson M, Beer EJ. Fed Proc 1959;18:418. Dubuisson D, Dennis SG. Pain 1977;4:161. Abbott FV, Melzack R. Brain Res 1982;251. Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. Pain 1992;51:5. Derardt R, Jougney S, Delevalcee F, Falhout M. Eur J Pharmacol 1980;51:17. Collier HDJ, Dinneen LC, Johnson CA, Schneider C. Br J Pharmacol 1968;32:295. Hunskaar S, Hole K. Pain 1987;30:103. Heapy CG, Jamieson A, Russel NJW. Br J Pharmacol 1987;90:164. Dickenson AH, Sullivian AF. Pain 1987;30:349. Dalal A, Tata M, Allegre G, Gekiere F, Bons N, Albe-Fessard D. Neuroscience 1999;94:217. Shibata M, Ohkubo T, Takahashi H, Inoki R. Pain 1989;38:347. Pathak D, Pathak K, Sigla AK. Fitoterapia 1991;62:371. Bittar M, de Sousa MM, Yunes R, Lento RA, Delle-Monache F, Cechinel-Filho V. Planta Med 2000;66:84. Duke JA. Handbook of biologically active phytochemicals and their activities. Boca, Raton, FL: CRC Press; 1992.
Evaluation of the antinociceptive activity of Ficus deltoidea aqueous extract M.R. Sulaiman a,⁎, M.K. Hussain a , Z.A. Zakaria b , M.N. Somchit a , S. Moin a , A.S. Mohamad a , D.A. Israf a a
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b Department of Pharmacology, Faculty of Pharmacy, Universiti Teknologi Mara, 40450 Shah Alam, Selangor, Malaysia Received 11 June 2008; accepted 18 June 2008 Available online 10 July 2008
Abstract The aqueous extract of Ficus deltoidea leaves was evaluated for possible antinociceptive activity in three models of nociception, namely, acetic acid-induced abdominal writhing, formalin and hot plate test. The results of the present study showed that intraperitoneal administration of the F. deltoidea leaves aqueous extract at the dose of 1, 50 and 100 mg/kg, 30 min prior to pain induction produced significant dose-dependent antinociceptive effect in all the models used, which indicating the presence of both central and peripherally mediated activities. Furthermore, the antinociceptive effect of the extract in the formalin and hot plate test was reversed by the non-selective opioid receptor antagonist naloxone suggesting that the endogenous opioid system is involved in its analgesic mechanism of action. Thus, the present results demonstrated that F. deltoidea leaves aqueous extract contains pharmacologically active constituents which possess antinociceptive activity justifying its popular therapeutic use in treating conditions associated with the painful conditions. © 2008 Elsevier B.V. All rights reserved. Keywords: Ficus deltoidea; Antinociceptive; Acetic acid-induced abdominal constriction test; Formalin test; Hot plate test
1. Introduction Ficus deltoidea is an epiphytic shrub which is native and widely distributed in several countries of the Southeast Asia. In Malaysia, it is commonly known as Mas cotek, serapat angin, telinga beruk and other names [1]. Different parts of the plant are used traditionally to treat various kinds of ailments.The fruits are chewed to relieve headache, toothache and cold; powdered root and leaves of the plant has been applied externally to wounds and sores, and around the joints for relief of rheumatism. Decoction or infusion from the whole plants is a well known traditional herbal drink for women after childbirth to help strengthen the uterus. Moreover it improves blood circulation, regain energy and it is believe to enhance sexual desire (personal communication from local people where the plant was collected). Besides being one of the popular herbs used in Malay traditional medicine, the pharmacological properties of this plant have not yet been studied, only ⁎ Corresponding author. Tel.: +6 03 89472603; fax: +6 03 89472585. E-mail address: [email protected] (M.R. Sulaiman). 0367-326X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.06.005
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one report of blood glucose lowering effect has been published [2]. The basis for the traditional use of this herb in the management of painful related conditions has not yet been scientifically verified to the best of our knowledge. The present study therefore, is aimed at investigating possible antinociceptive activity of the aqueous extract from the leaves of F. deltoidea and to explore its possible mechanism of action using chemical and thermal nociception models. 2. Experimental 2.1. Plant material The F. deltoideae fresh whole plant (Moraceae), collected from its natural habitat in Sepang, Selangor, Malaysia was identified by Mr. Shamsul Khamis. A voucher specimen (SK1510/2007) was deposited in the Phytomedicinal Herbarium, Institute of Biosciences, Universiti Putra Malaysia, Serdang, Selangor. 2.2. Aqueous extract The F. deltoideae leaves aqueous extract (FDA) was prepared as previously described [3]. Briefly, powdered dried leaves (100 g) infused in distilled water 60 °C for 2 days, filtered, and lyophilized yielding a dark brown colored semisolid mass (yields17.5%). The extract was kept at − 20 °C. 2.3. Animals Experiments were conducted using adult male Balb C mice (25–35 g) and adult male Sprague–Dawley rats (200–250 g), housed at 22± 2 °C under a 12-h light/12-h dark cycle and with access to food and water ad libitum. The animals were acclimatized to the laboratory for at least 1 h before testing and were used only once throughout the experiments. Experiments reported in this study were performed in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals [4]. 2.4. Preliminary phytochemical screening Phytochemical screening of the FDA was performed to detect the presence of different classes of constituents, such as alkaloids, flavonoids, saponins, steroids, terpenes and tannins [5,6]. 2.5. Antinociceptive tests 2.5.1. Acetic acid-induced abdominal writhing test The acetic acid-induced abdominal writhing test was performed according to procedures described previously [3,7]. Mice received the FDA (1, 50, and 100 mg/kg, i.p.) or normal saline 30 min prior to injection of 0.6% acetic acid (10 ml/kg, i.p.). The number of writhing was recorded for 30 min, starting 5 min after the injection of acetic acid. Acetylsalicylic acid (ASA, 100 mg/kg, i.p.) was administrated 30 min before the nociceptive agent. 2.5.2. Formalin test The test was performed according to Dubuisson and Dennis [8]. Briefly, 30 min after injections of the FDA (1, 50, and 100 mg/kg, i.p.) and ASA (100 mg/kg, i.p.) or normal saline, 50 μl of formaldehyde 2.5% (v/v in distilled water) was injected subcutaneously into the plantar surface of the left hind paw of the rats. The behavioral responses to nociception including biting, licking and scratching of the injected paw were noted and the time spent was recorded up to 30 min. The first 5 min was considered as early phase (neurogenic phase) and the period of 15–30 min as the late phase (inflammatory phase) of the nociceptive response. 2.5.3. Hot plate test The hot plate test was used to measure the response latencies according to the method described previously [3]. In these experiments, the hot plate (Ugo Basile, model-7280) was maintained at 55 ± 2 °C. Animals were placed into the perspex cylinder on the heated surface, and the time between placement and licking of the hind paws or jumping
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movements was recorded as response latency. The FDA (1, 50, 100 mg/kg, i.p.) and morphine (5 mg/kg, i.p.) were administered 30 min before the beginning of the experiment.Mice were observed before and at 30, 60, 120, 180, 240 and 300 min after substance administration. The cut-off time was 20 s. 2.5.4. Involvement of opioid receptors In order to verify the involvement of opioidergic system in the FDA-induced antinociception, separated groups of mice and rats (n = 6) were pre-treated with non-selective opioid receptor antagonist, naloxone (5 mg/kg, i.p), which was injected 15 min before the administration of the extract (100 mg/kg; i.p.) and morphine (5 mg/kg), and tested using the hot plate and the formalin testas described above. 2.6. Statistical analysis The statistical analyses were performed by ANOVA, followed by Dunnett's multiple comparison tests. The results were expressed as the mean ± S.E.M. to show variation in groups. Differences are considered significant when P ≤ 0.05. 3. Results and discussion The present study was conducted to assess the antinociceptive effectof the F. deltoidea, a medicinal claimed in Malay folk medicine for its analgesic property. It was demonstrated on chemical nociception in the test models of acetic acid-induced writhing, formalin-induced paw licking and on thermal nociception in hot plate test. These methods were selected such that both centrally and peripherally mediated effects were investigated. The acetic acid-induced abdominal constriction is believed to show the involvement of peripheral mechanisms, whereas the hot plate test is believed to show that of central mechanisms [9]. The formalin test is used to investigate both peripheral and central mechanisms [10]. Results of the present study demonstrated that the FDA possessed antinociceptive activity evident in all the nociceptive models, which suggestive of the presence of both central and peripherally mediated activities. In the acetic acid-induced abdominal constriction test, the results showed that the FDA extract (1, 50 and 100 mg/kg) potently and significantly reduced the number of abdominal writhing in a dose dependent manner with 50.6%, 70.4% and 88.0% of inhibition, respectively as compared to control animals (Table 1). The positive control group treated with acetylsalicylic acid (100 mg/kg) also manifested significantly reduction on the number of writhes (61.5%). It has been postulated that acetic acid acts indirectly by inducing the release of endogenous mediators, such as PGE2 (prostaglandin E2) and PGF2α in peritoneal fluids as well as lipooxygenase products, which stimulate the nociceptive neurons sensitive to NSAIDs [11,12]. Therefore, the result of the acetic acid-induced writhing strongly suggests that the mechanism of this extract may be linked partly to inhibition of lipooxygenase and/or cyclooxygenase in peripheral tissues, thereby reducing PGE2 synthesis and interfering with the mechanism of transduction in primary afferent nociceptor. In the formalin test which is sensitive for various classes of analgesic drugs [13], administration of the extract demonstrated significant dose dependent inhibition in both early (27.7%, 36.5%, and 68.5%) and late phase (31.9%, 44.5%, 72.5%) licking responses, at the tested doses of 1, 50 and 100 mg/kg, respectively (Table 2). Similarly, morphine produced marked inhibition of both the early phase (90.4%) and late phase (88.5%) of the formalin test. In contrast, the treatment of animals with ASA caused significant inhibition (73.3%) of the late phase, but not the early phase. It is also well known that the formalin test may involve sensorial C-fibers [14] in early phase and a combined Table 1 Effect of the F. deltoideae aqueous extract (FDA) on acetic acid-induced abdominal writhing test in mice Material Control (NaCl, 10 ml/kg, i.p.) FDA
Acetylsalicylic acid
Dose (mg/kg; i.p.)
No. of writhings
Inhibition (%)
1.0 50.0 100.0 100.0
62.0 ± 3.9 30.6 ± 2.7⁎ 18.4 ± 3.4⁎⁎ 7.4 ± 2.4⁎⁎ 24.0 ± 3.5⁎
– 50.6 70.4 88.0 61.5
Values are mean ± S.E.M.; (N = 10). ⁎ P b 0.05; ⁎⁎P b 0.01 significantly different from control (ANOVA followed by Dunnett's test).
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Table 2 Effect of the F. deltoideae aqueous extract (FDA) on formalin-induced pain in rats Material
Dose (mg/kg; i.p.)
Control (NaCl, 10 ml/kg, i.p.) FDA
Acetylsalicylic acid Morphine Morphine + Naloxone FDA + Naloxone
Total time spent in licking (s)
1.0 50.0 100.0 100.0 5 5+5 50 + 5
0–5 min
Inhibition (%)
15–30 min
Inhibition (%)
57.2 ± 11.0 41.3 ± 2.3⁎ 36.3 ± 2.3⁎ 18.0 ± 2.6⁎⁎ 51.8 ± 7.2 5.5 ± 0.6⁎⁎ 52.2 ± 2.3⁎# 50.6 ± 7.7⁎#
– 27.7 36.5 68.5 9.4 90.4 8.7 11.5
140.9 ± 8.4 96.0 ± 3.7⁎ 78.2 ± 4.6⁎ 38.7 ± 5.3⁎⁎ 37.6 ± 8.8⁎⁎ 16.2 ± 1.2⁎⁎ 129.6 ± 3.1⁎# 109.5 ± 9.7⁎#
– 31.9 44.5 72.5 73.3 88.5 8.0 22.3
Results are expressed as the mean ± S.E.M. in seconds (N = 10). ⁎P b 0.05; ⁎⁎P b 0.01 compared to the control group. # P b 0.05 compared to the group receiving appropriate drug/extract at the same dose without naloxone. (Dunnet's test).
process generated by peripheral inflammatory tissue and functional changes in the dorsal horn in late phase [15,16]. The centrally acting drugs such as narcotics inhibited both phases equally, while peripherally acting drugs only inhibited the second phase [17]. Inhibition of both phases of pain as observed with the extract in this study showed that they contain active analgesic principles acting both centrally and peripherally. The central analgesic effect of the aqueous extract may be supported by the results recorded in the hot plate test which is a selective method able to screen centrally acting opiate analgesic drugs [9]. It was demonstrated that intraperitoneal administration of the extract (1, 50 and 100 mg/kg) exerts significant prolonged in the response latency time to the heat stimulus (Table 3). This effect begun early at 30 min after administration of aqueous extract and persists until the following fifth hour. As expected, morphine (5 mg/kg, i.p.) significantly increased the latency time to the nociceptive response compared with control group. It is also interesting to note that, pre-treatment with a non-selective opioid receptor antagonist, naloxone significantly reversed the antinociceptive effect of FDA extract and morphine in both phases of the formalin test as well as in the hot plate test, compared to extract and morphine alone (Tables 2 and 3). These results suggest that activation of central opioid receptors are involved in the antinociceptive action of the FDA extract. Preliminary phytochemical screening carried out on this species showed the presence of numerous constituents' classes, such as flavonoids, saponins, tannins, steroids and terpenes. Since several flavonoids and tannins isolated from medicinal plants have been discovered for their significant antinociceptive and/or anti-inflammatory activity [18–20]. It is, therefore, possible that the antinociceptive effects observed with this extract in the present study may be attributable to its flavonoids and tannins component. In addition, the preliminary acute oral toxicity test obtained with this plant showed no death occurred, even at the highest dose of FDA (5 g/kg), indicating it may have a reasonable safety margin with regards to acute toxicity (data not shown). This further justifying its wide application in treatment of pain in various disease conditions and lack of any reported side effects with the traditional use of this plant. Table 3 Effect of the F. deltoideae aqueous extract (FDA) on the hot plate test in mice Material
Control (NaCl) FDA
FDA + Naloxone Morphine Morphine + Naloxone
Dose (mg/kg)
1 50 100 100 + 5 5 5+5
Latency time 0
30
60
120
180
240
300
6.4 ± 1.2 7.2 ± 0.5 6.5 ± 0.3 6.9 ± 1.5 6.6 ± 0.3 7.9 ± 1.6 6.5 ± 1.1
7.3 ± 0.6 8.5 ± 1.1⁎ 9.4 ± 0.3⁎ 11.9 ± 0.6⁎ 7.54 ± 0.3# 17.1 ± 0.3⁎ 8.4 ± 2.2#
7.8 ± 0.6 9.4 ± 0.2⁎ 11.0 ± 0.6⁎ 11.9 ± 1.6⁎ 6.89 ± 0.3# 18.1 ± 0.6⁎ 8.3 ± 1.3#
7.5 ± 1.1 9.6 ± 0.3⁎ 12.7 ± 0.5⁎ 14.8 ± 0.5⁎ 7.17 ± 0.7# 19.0 ± 2.2⁎ 8.0 ± 1.5#
6.9 ± 1.6 8.4 ± 0.6⁎ 11.5 ± 1.2⁎ 13.9 ± 0.3⁎ 7.53 ± 2.3# 16.8 ± 0.3⁎ 8.5 ± 0.3#
7.4 ± 0.5 9.5 ± 0.3⁎ 12.9 ± 0.6⁎ 14.7 ± 1.3⁎ 7.67 ± 0.6# 15.4 ± 0.4⁎ 10.3 ± 1.6#
6.5 ± 0.3 7.3 ± 0.3 10.0 ± 1.3⁎ 11.2 ± 0.3⁎ 6.24 ± 1.3# 14.2 ± 0.3⁎ 7.5 ± 0.3#
Results are expressed as the mean ± S.E.M. in seconds (N = 8). ⁎P b 0.05 compared to the control group. # P b 0.05 compared to the group receiving appropriate drug/extract at the same dose without naloxone (Dunnet’s test).
M.R. Sulaiman et al. / Fitoterapia 79 (2008) 557–561
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In conclusion, the present study demonstrated that the aquoues extract of FD possess significant antinociceptive effects in laboratory animals at the doses investigated. The results seems to support the traditional use of this plant in some painful conditions and also suggest the presence of biologically active principles which may be worth further investigation and elucidation. Further studies are in fact currently under way to isolate and characterize the active principle(s) of the crude extract. Acknowledgement The authors would like to thank the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for providing the necessary support for the study. This research was supported by a Research University Grant Scheme 2007 (04/01/07/0093RU) from the Universiti Putra Malaysia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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