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Antinociceptive effect of Hyptis pectinata leaves extracts
Fitoterapia 77 (2006) 439 – 442 www.elsevier.com/locate/fitote
Antinociceptive effect of Hyptis pectinata leaves extracts Ana C.C.D. Lisboa ⁎, Iderjane C.M. Mello, Rogeria S. Nunes, Marquês A. dos Santos, Angelo R. Antoniolli, Rosilene M. Marçal, Sócrates C. de H. Cavalcanti Physiology Department, Federal University of Sergipe, São Cristovão, Sergipe, Brazil Received 5 January 2006; accepted 12 June 2006 Available online 6 July 2006
Abstract Oral administration of hexanes, chloroform, and ethyl acetate extracts of the leaves of Hyptis pectinata significantly reduced the number of writhing induced by acetic acid and increased the response to thermal stimuli in hot-plate test. Such effect was completely reversed by the opioid antagonist naloxone. © 2006 Elsevier B.V. All rights reserved. Keywords: Hyptis pectinata; Analgesy; Naloxone
1. Introduction Hyptis pectinata L., Poit (Lamiaceae) is a common bush known as Sambacaita or Canudinho in Sergipe State (Brazil) widely used to combat bacterial infections, inflammation, cancer, and pain. In a previous study, we described the analgesic effect of the aqueous extract of the H. pectinata leaves which exerts marked analgesic action in mice [1]. In view of this fact, it was of our interest to evaluate the hexanes, chloroform, and ethyl acetate extracts from H. pectinata leaves. General ethnomedicinal information of this plant describes its use in rhinopharingites, nasal congestion, skin disesases, fever, bacterial infections, and pain treatment [2,3]. Investigations of the H. pectinata methanol extract have shown antimicrobial activity [3] while the aqueous extract enhanced liver hepatic regeneration [4]. The essential oil of H. pectinata was analyzed by CG/MS. Thirty-three compounds, with quantitative predominance of monoterpenes, were detected in the oil of its leaves [2]. The antiseptic property has been attributed to high percentages of thymol [2]. The essential oil from H. pectinata collected in Cameroon contained germacrene D and βcaryophyllene as major constituents [5].
⁎ Corresponding author. Caixa Postal 52, CEP 49.001-970, Aracaju, Sergipe, Brazil. Tel.: +55 79 3212 6641; fax: +55 79 3243 7457. E-mail address: [email protected] (A.C.C.D. Lisboa). URL: http://www.ufs.br (A.C.C.D. Lisboa). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.001
440
A.C.C.D. Lisboa et al. / Fitoterapia 77 (2006) 439–442
Bioassay-guided fractionation of the chloroform extract of H. pectinata led to the isolation of pyrones and pectinolides. The isolated pectinolides were effective against bacteria and tumor cell lines [6–8]. 2. Experimental 2.1. Plant H. pectinata leaves, collected in August 2003 in Aracaju, were identified by the Biologist Gilvane Viana Souza. A voucher specimen (No. ASE2626) was deposited in the Herbarium of the Biology Department , Federal University of Sergipe, São Cristóvão, Sergipe, Brazil. 2.2. Preparation of extracts Dried powdered leaves (500 g) were macerated in MeOH at r.t. The solvent was removed in vacuo to give a dark green powder (yield: 13%). The dry dark green powder was Soxhlet partitioned to give hexane (3.2%), chloroform (4.3%), and ethyl acetate (0.7%) extracts. 2.3. Animals Male and female Swiss mice (20–35 g) were used. The animals had free access to pellet diet (Purina chow) and tap water. Animal Care and Use Committee of our Institute approved the experimental protocols in accordance with the ethical guidelines for the investigation of experimental pain in conscious animals [9]. 2.4. Writhing test Writhing activity was evaluated according Koster et al. [10]. Nine animals were included in each group. The number of writhes/mouse was counted during a 20 min period, starting 10 min after i.p. administration of 0.6% acetic acid (10 ml/kg). Water or extracts (100, 200, and 400 mg/kg) were administered orally 1 h before the nociceptive agent to overnight fasted animals. Morphine (2.5 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) were used as standard and administered 1 h before administration of acetic acid. 2.5. Hot-plate test [11] The experiment started 60 min (time zero) after oral administration of hexanes, chloroform, and ethyl acetate extracts (100, 200, and 400 mg/kg). Eight mice were placed on the hot-plate (55 ± 0.5 °C) and the time elapsed to the moment when they licked their hind paws was recorded (hereafter referred to as latency). This procedure was repeated at 0, +15, +30, and +60 min. In order to avoid damage to the paws of the animals, the time standing on the plate was limited to 30 s. The opioid receptor antagonist naloxone (5 mg/kg, i.p.) was tested along with the effective extract doses. Saline (i.p., control group for morphine and naloxone), naloxone (5 mg/kg, i.p), morphine (7.5 mg/kg, i.p.), and morphine plus naloxone (the same doses) were used as the reference drugs and administered 45 min before the first measure. 2.6. Statistical analysis Mean values and SEM were calculated for all results. The results were analyzed using ANOVA analysis of variance, followed by Dunnett's test when appropriate. A significance level of 5% was set for all analyses. 3. Results and discussion The results are reported in Tables 1 and 2. Hexanes, chloroform, and ethyl acetate extracts (100, 200, and 400 mg/ kg, p.o.) of H. pectinata leaves significantly reduced the number of writhing induced by acetic acid 0.6%. Moreover, all fractions significantly increased the hindpaw lick latency on the hot-plate test. Since it is believed that this test is
A.C.C.D. Lisboa et al. / Fitoterapia 77 (2006) 439–442
441
Table 1 Effects of hexanes, ethyl acetate, and chloroform extracts from H. pectinata leaves on the acetic acid-induced writhing in mice Extracts
Dose (mg/kg)
No. of writhings (Mean ± S.E.M.)
Inhibition (%)
Control (water; p.o.)
– 100 200 400 – 100 200 400 – 100 200 400 – 2.5 10
22.55 ± 1.65 9.54 ± 1.27*** 3.18 ± 0.77*** 2.00 ± 0.63*** 24.78 ± 1.01 4.11 ± 0.94*** 4.44 ± 0.88*** 2.67 ± 0.76*** 23.00 ± 3.70 8.37 ± 1.27*** 6.25 ± 1.37*** 1.85 ± 0.81*** 30.33 ± 3.12 1.33 ± 0.50*** 2.67 ± 0.91***
– 61.8a 87.3a 92.0a – 83.6a 82.4a 89.2a – 66.5a 75.0a 92.1a
Hexane extract (p.o.) Control (water; p.o.) Chloroform extract (p.o.) Control (water; p.o.) Ethyl acetate extract (p.o.) Control (saline i.p.) Morphine (i.p.) Indometacin (i.p.)
95.6b 91.2b
Values are expressed as mean ± S.E.M. N = 9. ⁎⁎⁎P b 0.001 vs control (ANOVA, followed by Dunnett's). a % Inhibition compared to water control. b % Inhibition compared to saline control.
sensitive to drugs acting supraspinally [12], our results suggest that all fractions act on this site. In order to better evaluate this aspect the non-selective opioid antagonist naloxone (5 mg/kg, i.p.) was co-administrated with chloroform (200 mg/kg), ethyl acetate (100 mg/kg), and hexanes extracts (100 mg/kg). Since naloxone reversed the antinociceptive effect, we can suggest that those effects are probably related to the opioid receptors activation.
Table 2 Effects of hexanes, ethyl acetate, and chloroform extracts from H. pectinata leaves on the hot-plate test in mice Extracts
Control (water) Chloroform extract
Chloroform extract + Naloxone Control (water) Hexane extract Hexane extract + Naloxone Control (water) Ethyl acetate extract Ethyl acetate + Naloxone Control (saline) Morphine Morphine + Naloxone Naloxone
Dose (mg/kg) – 100 200 400 200 + 5 – 100 200 400 400 + 5 – 100 200 400 100 + 5 – 7.5 7.5 + 5 5
Values are expressed as mean ± S.E.M. N = 08. ⁎ P b 0.05 vs control (ANOVA, followed by Dunnett's). ⁎⁎ P b 0.001 vs control (ANOVA, followed by Dunnett's). ⁎⁎⁎ P b 0.01 vs control (ANOVA, followed by Dunnett's).
Hindpaw lick latency time (s) 0
15
30
60
7.63 ± 0.38 8.31 ± 0.59 8.19 ± 0.72 8.40 ± 0.82 9.91 ± 1.43 9.87 ± 1.33 11.11 ± 0.75 13.32 ± 0.84⁎ 13.72 ± 0.88⁎ 11.88 ± 0.55 8.87 ± 0.46 12.30 ± 1.45⁎⁎ 9.31 ± 0.88 10.50 ± 0.58 11.09 ± 0.50 8.46 ± 0.51 30.0 ± 0.00⁎⁎ 10.98 ± 0.52 7.12 ± 0.36
8.01 ± 0.90 10.18 ± 0.84 12.57 ± 1.20⁎ 10.70 ± 1.08 10.13 ± 1.24 10.52 ± 0.89 13.97 ± 0.76⁎ 13.76 ± 0.87 15.45 ± 1.30⁎⁎ 11.50 ± 0.93 9.64 ± 0.65 10.89 ± 1.12 11.74 ± 0.59 14.32 ± 0.55⁎⁎ 11.04 ± 0.69 8.49 ± 0.16 30.0 ± 0.00⁎⁎ 10.82 ± 0.92 8.89 ± 0.97
7.74 ± 0.55 7.78 ± 0.73 10.78 ± 0.44 8.98 ± 1.01 9.51 ± 1.63 12.90 ± 1.52 13.72 ± 1.40 13.85 ± 1.24 16.82 ± 1.11 10.09 ± 1.25 9.79 ± 0.54 10.74 ± 1.09 9.94 ± 0.96 10.83 ± 0.84 11.83 ± 0.74 8.64 ± 0.66 30.0 ± 0.00⁎⁎ 11.13 ± 0.77 8.39 ± 0.67
9.18 ± 0.73 9.95 ± 1.23 14.28 ± 1.29⁎ 11.46 ± 1.44 11.53 ± 0.65 13.42 ± 1.29 14.27 ± 1.02 14.47 ± 1.56 15.45 ± 1.17 11.98 ± 1.15 8.80 ± 0.83 12.03 ± 0.98⁎ 11.44 ± 0.68 12.39 ± 0.90⁎ 10.10 ± 0.85 7.45 ± 0.71 30.0 ± 0.00⁎⁎⁎ 10.12 ± 0.63 9.07 ± 0.74
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In conclusion, H. pectinata extracts have significant antinociceptive property with an opioid-like effect and supraspinal site of action. Acknowledgements The authors wish to acknowledge CNPq and FAP-SE for the financial support. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
Bispo MD, Mourao RHV, Franzotti EM, Bomfim KBR, Arrigoni-Blank MD, Moreno MPN, et al. J Ethnopharmacol 2001;76:81. Malan K, Pelissier Y, Marion C, Blaise A, Bessiere JM. Planta Med 1988:531. Rojas A, Hernandez L, Peredamiranda R, Mata R. J Ethnopharmacol 1992;35:275. Melo GB, Silva RL, Melo VA, Lima SO, Antoniolli AR, Castro-E-Silva T, et al. Dig Dis Sci 2005;50:949. Tchoumbougnang F, Zollo PHA, Boyom FF, Nyegue MA, Bessiere JM, Menut C. Flavour Fragr J 2005;20:340. Fragoso-Serrano M, Gibbons S, Pereda-Miranda R. Planta Med 2005;71:278. Peredamiranda R, Hernandez L, Ibarra P. Abstr Pap Am Chem Soc 1991;202:60. Peredamiranda R, Hernandez L, Villavicencio MJ, Novelo M, Ibarra P, Chai HY, et al. J Nat Prod 1993;56:583. Zimmermann M. Pain 1983;16:109. Koster R, Anderson M, Debeer EJ. Fed Proc 1959;18:412. Ankier SI. Eur J Pharmacol 1974;27:1. Yaksh TL, Rudy TA. J Pharmacol Exp Ther 1977;202:411.
Antinociceptive effect of Hyptis pectinata leaves extracts Ana C.C.D. Lisboa ⁎, Iderjane C.M. Mello, Rogeria S. Nunes, Marquês A. dos Santos, Angelo R. Antoniolli, Rosilene M. Marçal, Sócrates C. de H. Cavalcanti Physiology Department, Federal University of Sergipe, São Cristovão, Sergipe, Brazil Received 5 January 2006; accepted 12 June 2006 Available online 6 July 2006
Abstract Oral administration of hexanes, chloroform, and ethyl acetate extracts of the leaves of Hyptis pectinata significantly reduced the number of writhing induced by acetic acid and increased the response to thermal stimuli in hot-plate test. Such effect was completely reversed by the opioid antagonist naloxone. © 2006 Elsevier B.V. All rights reserved. Keywords: Hyptis pectinata; Analgesy; Naloxone
1. Introduction Hyptis pectinata L., Poit (Lamiaceae) is a common bush known as Sambacaita or Canudinho in Sergipe State (Brazil) widely used to combat bacterial infections, inflammation, cancer, and pain. In a previous study, we described the analgesic effect of the aqueous extract of the H. pectinata leaves which exerts marked analgesic action in mice [1]. In view of this fact, it was of our interest to evaluate the hexanes, chloroform, and ethyl acetate extracts from H. pectinata leaves. General ethnomedicinal information of this plant describes its use in rhinopharingites, nasal congestion, skin disesases, fever, bacterial infections, and pain treatment [2,3]. Investigations of the H. pectinata methanol extract have shown antimicrobial activity [3] while the aqueous extract enhanced liver hepatic regeneration [4]. The essential oil of H. pectinata was analyzed by CG/MS. Thirty-three compounds, with quantitative predominance of monoterpenes, were detected in the oil of its leaves [2]. The antiseptic property has been attributed to high percentages of thymol [2]. The essential oil from H. pectinata collected in Cameroon contained germacrene D and βcaryophyllene as major constituents [5].
⁎ Corresponding author. Caixa Postal 52, CEP 49.001-970, Aracaju, Sergipe, Brazil. Tel.: +55 79 3212 6641; fax: +55 79 3243 7457. E-mail address: [email protected] (A.C.C.D. Lisboa). URL: http://www.ufs.br (A.C.C.D. Lisboa). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.001
440
A.C.C.D. Lisboa et al. / Fitoterapia 77 (2006) 439–442
Bioassay-guided fractionation of the chloroform extract of H. pectinata led to the isolation of pyrones and pectinolides. The isolated pectinolides were effective against bacteria and tumor cell lines [6–8]. 2. Experimental 2.1. Plant H. pectinata leaves, collected in August 2003 in Aracaju, were identified by the Biologist Gilvane Viana Souza. A voucher specimen (No. ASE2626) was deposited in the Herbarium of the Biology Department , Federal University of Sergipe, São Cristóvão, Sergipe, Brazil. 2.2. Preparation of extracts Dried powdered leaves (500 g) were macerated in MeOH at r.t. The solvent was removed in vacuo to give a dark green powder (yield: 13%). The dry dark green powder was Soxhlet partitioned to give hexane (3.2%), chloroform (4.3%), and ethyl acetate (0.7%) extracts. 2.3. Animals Male and female Swiss mice (20–35 g) were used. The animals had free access to pellet diet (Purina chow) and tap water. Animal Care and Use Committee of our Institute approved the experimental protocols in accordance with the ethical guidelines for the investigation of experimental pain in conscious animals [9]. 2.4. Writhing test Writhing activity was evaluated according Koster et al. [10]. Nine animals were included in each group. The number of writhes/mouse was counted during a 20 min period, starting 10 min after i.p. administration of 0.6% acetic acid (10 ml/kg). Water or extracts (100, 200, and 400 mg/kg) were administered orally 1 h before the nociceptive agent to overnight fasted animals. Morphine (2.5 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) were used as standard and administered 1 h before administration of acetic acid. 2.5. Hot-plate test [11] The experiment started 60 min (time zero) after oral administration of hexanes, chloroform, and ethyl acetate extracts (100, 200, and 400 mg/kg). Eight mice were placed on the hot-plate (55 ± 0.5 °C) and the time elapsed to the moment when they licked their hind paws was recorded (hereafter referred to as latency). This procedure was repeated at 0, +15, +30, and +60 min. In order to avoid damage to the paws of the animals, the time standing on the plate was limited to 30 s. The opioid receptor antagonist naloxone (5 mg/kg, i.p.) was tested along with the effective extract doses. Saline (i.p., control group for morphine and naloxone), naloxone (5 mg/kg, i.p), morphine (7.5 mg/kg, i.p.), and morphine plus naloxone (the same doses) were used as the reference drugs and administered 45 min before the first measure. 2.6. Statistical analysis Mean values and SEM were calculated for all results. The results were analyzed using ANOVA analysis of variance, followed by Dunnett's test when appropriate. A significance level of 5% was set for all analyses. 3. Results and discussion The results are reported in Tables 1 and 2. Hexanes, chloroform, and ethyl acetate extracts (100, 200, and 400 mg/ kg, p.o.) of H. pectinata leaves significantly reduced the number of writhing induced by acetic acid 0.6%. Moreover, all fractions significantly increased the hindpaw lick latency on the hot-plate test. Since it is believed that this test is
A.C.C.D. Lisboa et al. / Fitoterapia 77 (2006) 439–442
441
Table 1 Effects of hexanes, ethyl acetate, and chloroform extracts from H. pectinata leaves on the acetic acid-induced writhing in mice Extracts
Dose (mg/kg)
No. of writhings (Mean ± S.E.M.)
Inhibition (%)
Control (water; p.o.)
– 100 200 400 – 100 200 400 – 100 200 400 – 2.5 10
22.55 ± 1.65 9.54 ± 1.27*** 3.18 ± 0.77*** 2.00 ± 0.63*** 24.78 ± 1.01 4.11 ± 0.94*** 4.44 ± 0.88*** 2.67 ± 0.76*** 23.00 ± 3.70 8.37 ± 1.27*** 6.25 ± 1.37*** 1.85 ± 0.81*** 30.33 ± 3.12 1.33 ± 0.50*** 2.67 ± 0.91***
– 61.8a 87.3a 92.0a – 83.6a 82.4a 89.2a – 66.5a 75.0a 92.1a
Hexane extract (p.o.) Control (water; p.o.) Chloroform extract (p.o.) Control (water; p.o.) Ethyl acetate extract (p.o.) Control (saline i.p.) Morphine (i.p.) Indometacin (i.p.)
95.6b 91.2b
Values are expressed as mean ± S.E.M. N = 9. ⁎⁎⁎P b 0.001 vs control (ANOVA, followed by Dunnett's). a % Inhibition compared to water control. b % Inhibition compared to saline control.
sensitive to drugs acting supraspinally [12], our results suggest that all fractions act on this site. In order to better evaluate this aspect the non-selective opioid antagonist naloxone (5 mg/kg, i.p.) was co-administrated with chloroform (200 mg/kg), ethyl acetate (100 mg/kg), and hexanes extracts (100 mg/kg). Since naloxone reversed the antinociceptive effect, we can suggest that those effects are probably related to the opioid receptors activation.
Table 2 Effects of hexanes, ethyl acetate, and chloroform extracts from H. pectinata leaves on the hot-plate test in mice Extracts
Control (water) Chloroform extract
Chloroform extract + Naloxone Control (water) Hexane extract Hexane extract + Naloxone Control (water) Ethyl acetate extract Ethyl acetate + Naloxone Control (saline) Morphine Morphine + Naloxone Naloxone
Dose (mg/kg) – 100 200 400 200 + 5 – 100 200 400 400 + 5 – 100 200 400 100 + 5 – 7.5 7.5 + 5 5
Values are expressed as mean ± S.E.M. N = 08. ⁎ P b 0.05 vs control (ANOVA, followed by Dunnett's). ⁎⁎ P b 0.001 vs control (ANOVA, followed by Dunnett's). ⁎⁎⁎ P b 0.01 vs control (ANOVA, followed by Dunnett's).
Hindpaw lick latency time (s) 0
15
30
60
7.63 ± 0.38 8.31 ± 0.59 8.19 ± 0.72 8.40 ± 0.82 9.91 ± 1.43 9.87 ± 1.33 11.11 ± 0.75 13.32 ± 0.84⁎ 13.72 ± 0.88⁎ 11.88 ± 0.55 8.87 ± 0.46 12.30 ± 1.45⁎⁎ 9.31 ± 0.88 10.50 ± 0.58 11.09 ± 0.50 8.46 ± 0.51 30.0 ± 0.00⁎⁎ 10.98 ± 0.52 7.12 ± 0.36
8.01 ± 0.90 10.18 ± 0.84 12.57 ± 1.20⁎ 10.70 ± 1.08 10.13 ± 1.24 10.52 ± 0.89 13.97 ± 0.76⁎ 13.76 ± 0.87 15.45 ± 1.30⁎⁎ 11.50 ± 0.93 9.64 ± 0.65 10.89 ± 1.12 11.74 ± 0.59 14.32 ± 0.55⁎⁎ 11.04 ± 0.69 8.49 ± 0.16 30.0 ± 0.00⁎⁎ 10.82 ± 0.92 8.89 ± 0.97
7.74 ± 0.55 7.78 ± 0.73 10.78 ± 0.44 8.98 ± 1.01 9.51 ± 1.63 12.90 ± 1.52 13.72 ± 1.40 13.85 ± 1.24 16.82 ± 1.11 10.09 ± 1.25 9.79 ± 0.54 10.74 ± 1.09 9.94 ± 0.96 10.83 ± 0.84 11.83 ± 0.74 8.64 ± 0.66 30.0 ± 0.00⁎⁎ 11.13 ± 0.77 8.39 ± 0.67
9.18 ± 0.73 9.95 ± 1.23 14.28 ± 1.29⁎ 11.46 ± 1.44 11.53 ± 0.65 13.42 ± 1.29 14.27 ± 1.02 14.47 ± 1.56 15.45 ± 1.17 11.98 ± 1.15 8.80 ± 0.83 12.03 ± 0.98⁎ 11.44 ± 0.68 12.39 ± 0.90⁎ 10.10 ± 0.85 7.45 ± 0.71 30.0 ± 0.00⁎⁎⁎ 10.12 ± 0.63 9.07 ± 0.74
442
A.C.C.D. Lisboa et al. / Fitoterapia 77 (2006) 439–442
In conclusion, H. pectinata extracts have significant antinociceptive property with an opioid-like effect and supraspinal site of action. Acknowledgements The authors wish to acknowledge CNPq and FAP-SE for the financial support. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
Bispo MD, Mourao RHV, Franzotti EM, Bomfim KBR, Arrigoni-Blank MD, Moreno MPN, et al. J Ethnopharmacol 2001;76:81. Malan K, Pelissier Y, Marion C, Blaise A, Bessiere JM. Planta Med 1988:531. Rojas A, Hernandez L, Peredamiranda R, Mata R. J Ethnopharmacol 1992;35:275. Melo GB, Silva RL, Melo VA, Lima SO, Antoniolli AR, Castro-E-Silva T, et al. Dig Dis Sci 2005;50:949. Tchoumbougnang F, Zollo PHA, Boyom FF, Nyegue MA, Bessiere JM, Menut C. Flavour Fragr J 2005;20:340. Fragoso-Serrano M, Gibbons S, Pereda-Miranda R. Planta Med 2005;71:278. Peredamiranda R, Hernandez L, Ibarra P. Abstr Pap Am Chem Soc 1991;202:60. Peredamiranda R, Hernandez L, Villavicencio MJ, Novelo M, Ibarra P, Chai HY, et al. J Nat Prod 1993;56:583. Zimmermann M. Pain 1983;16:109. Koster R, Anderson M, Debeer EJ. Fed Proc 1959;18:412. Ankier SI. Eur J Pharmacol 1974;27:1. Yaksh TL, Rudy TA. J Pharmacol Exp Ther 1977;202:411.