Anticonvulsant Activity of Extracts of Plectranthus barbatus Leaves in Mice

Abstract

Plectranthus barbatus is a medicinal plant used to treat a wide range of disorders including seizure. However, the anticonvulsant activity of this plant has not been studied in depth. We therefore sought to evaluate the anticonvulsant activity of a hydroalcoholic extract of P. barbatus leaves on seizures induced by strychnine sulphate (2.0 mg/kg) and pilocarpine (600 mg/kg) in mice. The extract was administered orally at 1, 10, 30, and 100 mg/kg. We report that the P. barbatus extract had marked anticonvulsant activity against strychnine-induced convulsions, but was quite ineffective against pilocarpine-induced convulsions. Further experiments will be required to identify the active molecules(s) and their mechanism(s) of action.

1. Introduction

Plectranthus barbatus Andr. is a perennial shrub that is thought to have originated in Africa and is used as a medicinal plant to treat a wide range of disorders. The plant is also known by the synonyms Plectranthus forskohlii Briq, Plectranthus forskalaei Willd., Plectranthus kilimandschari (Gürke) H.L. Maass., Plectranthus grandis (Cramer) R.H. Willemse, Coleus forskohlii Briq., Coleus kilimandschari Gürke ex Engl., Coleus coerulescens Gürke, and Coleus barbatus (Andr.) Benth [1]. The medicinal uses of P. barbatus include digestive, respiratory, circulatory, nervous disorders [1] and infections [2] and has been reported to have abortifacient, contraceptive [3], and antipain [4] properties. The plant is also used to treat gastritis and intestinal spasms [5], nausea [6], stomach ache, and as a purgative [7]. Uses in respiratory disorders include the relief of colds [8], cough, [4] and bronchitis [9], and in the circulatory system uses include myalgia, angina, and hypertension [4].

P. barbatus is also used for the treatment of epilepsy and convulsions [10]; however, only limited data are available concerning the anticonvulsant activity of this plant. The present work was undertaken to evaluate the anticonvulsant activity of an extract of Plectranthus barbatus leaves.

2. Methods

2.1. Plant Extract Preparation

Fresh leaves of cultivated Plectranthus barbatus were obtained from the Medicinal and Toxic Plants Garden, Sector of Seedlings Production, Universidade Federal Rural do Semi-Árido—UFERSA, Mossoró, RN, Brazil. A reference specimen is deposited in the Prisco Bezerra Herbarium at the Universidade Federal do Ceará, Fortaleza, CE, Brazil, under number 24408. Leaves were air-dried at 35°C, pulverized, and the powder was extracted with ethanol : water (20 : 80), filtered, and dried by evaporation. The dry extract was dissolved in distillated water at concentration of 50 mg dried P. barbatus solids per ml.

2.2. Animals

Swiss albino male mice, weighing between 25 to 30 g, two months old, were kept under controlled conditions (21–25°C on a 12 h : 12 h light : dark cycle). Animals were maintained on a standard mouse diet; before the experiments they were fasted overnight with water ad libitum.

Ethical procedures were based on the Brazilian law 6638 (May 8, 1979) “Normas para Prática Didático-Científica da Vivissecção de Animais” and “Ethical Principles for Use of Experimental Animals” from Colégio Brasileiro de Experimentação Animal (COBEA), Brazil, which are in accordance with the “European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes” (Strasbourg, March 18, 1986).

2.3. Evaluation of Anticonvulsant Activity

For each seizure-inducing drug tested, 40 mice were divided into five groups of eight animals; P. barbatus extract was administered by oral gavage at doses of 0, 1, 10, 30, and 100 mg/kg. Thirty minutes after administration, each mouse was injected intraperitoneally with strychnine (2.0 mg/kg) or with pilocarpine (600 mg/kg). The doses of used drugs were defined in a pilot study. The latency to first convulsion and the latency and percentage mortality were recorded for a period of 30 min. Animals surviving more than 30 min were considered to be protected.

2.4. Statistical Analysis

Results are expressed as means ± standard error of the mean (SEM). Comparisons between the averages of series of values were performed by ANOVA followed by Dunnett’s multiple comparisons test. Data analysis employed Graphpad INSTAT version 2.0 software; statistical significance was set at .

3. Results

P. barbatus extract at doses of 1 and 100 mg/kg significantly delayed the onset of strychnine-induced seizures (Table 1). At a dose of 1 mg/kg, there was a significant increase in the latency to death, but the extract failed to protect the animals against drug-induced death. At a dose of 10 mg/kg, there was no significant difference in time before onset of seizure or death latency. However, P. barbatus extract at dose of 30 mg/kg protected 12.5% (1/8) of treated animals; although time to seizure onset and latency to death were increased by extract administration the difference failed to achieve statistical significance . At 100 mg/kg, all treated animals (8/8) were now protected against drug-induced death .

For pilocarpine-induced seizures, all P. barbatus extract doses (1, 10, 30, and 100 mg/kg) significantly delayed seizure onset, and doses of 1, 10, and 100 mg/kg significantly increased the latency to death. However, no significant retardation of death latency at a dose of 30 mg/kg was seen compared to the control group, and no dose protected animals from convulsions or death (Table 2).

4. Discussion

We report that a hydroalcoholic extract of leaves of Plectranthus barbatus exerts anticonvulsant effects against both strychnine- and pilocarpine-induced seizures. All tested doses of P. barbatus extract retarded pilocarpine-induced seizures but failed to protect against death. By contrast, no seizure activity and no death were observed in strychnine-treated animals pretreated with the highest tested dose of P. barbatus extract (100 mg/kg).

The mechanism underlying strychnine-induced seizures is thought to involve direct antagonism of strychnine-sensitive glycine receptors not only in higher brain areas but also in the spinal cord and brainstem, thereby abrogating spinal reflexes and causing motor disturbance, increased muscle tone, hyperactivity of sensory, visual and acoustic perception, tonic convulsions, and death through respiratory or spinal paralysis or by cardiac arrest [11]. Our results demonstrate that strychnine-induced seizures are partly suppressed by P. barbatus treatment.

Pilocarpine is a nonspecific muscarinic acetylcholine receptor agonist; it has been suggested that cerebral structures with a high density of muscarinic receptors are likely to represent the sites of origin of acute pilocarpine seizures [12]. Pilocarpine administration to rodents leads to repetitive limbic seizures and status epilepticus, replicating several features of human temporal lobe epilepsy [13]. It was previously suggested that extracts of P. amboinicus might antagonize cholinergic action at muscarinic receptors [14]. On the other hand, P. barbatus extracts were previously reported to inhibit acetylcholinesterase activity [15]. In the present work, the administration of P. barbatus extracts failed to inhibit pilocarpine-induced seizures, even though it retarded the onset of the seizures. Thus, it appears likely that P. barbatus extract presented slight antagonist action in the muscarinic acetylcholine receptors.

Phytochemical studies on P. barbatus have revealed numerous bioactive compounds: notably diterpenoids including forskolin [16], plectranthone J, plectrin, (16S)-coleon E, coleon F, (16R)-plectrinone A, plectrinone B [17], cyclobarbatusin, barbatusin, and 7β-acetyl-12-desacetoxicyclobarbatusin [18]. Of these, forskolin is a promising candidate for the anticonvulsant activity of P. barbatus because this molecule was shown to suppress seizures induced by pentylenetetrazol [19]. Forskolin activates adenylate cyclase and increases intracellular cAMP levels. It was previously reported that forskolin can elicit membrane depolarization in medium spiny neurons of the mouse nucleus accumbens; this was mediated by cAMP stimulation of inward rectifier K⁺ currents [20]. Forskolin increases the amplitude of glycinergic miniature inhibitory postsynaptic currents in the presence of low concentrations of extracellular glycine in isolated rat substantia gelatinosa neurons [21].

In addition to direct effects on ion channels, forskolin treatment is likely to exert neuroprotective effects by increasing cellular levels of neurotrophin-3 (NT-3) [22], a member of the neurotrophin family of neurotrophic factors that support cell survival, axonal growth, and neuronal plasticity [23, 24]. Brain injury decreases NT-3 synthesis in the hippocampus [25], whereas NT-3 supplementation can protect neurons against excitotoxic insult [26]. Seizures induced by kainic acid [27] or pilocarpine [28] injection decreased NT-3 expression in the brain of mice, whereas intraventricular infusion of NT-3 in rats inhibited kindling epileptogenesis, mossy fiber sprouting, and increases in hilar area [29], arguing that NT-3 upregulation by forskolin is likely to inhibit seizure development and seizure-related synaptic reorganization.

5. Conclusion

In conclusion, P. barbatus extract was found to have marked anticonvulsant activity against strychnine-induced convulsions, but was quite ineffective against pilocarpine-induced convulsions. Although forskolin is a promising candidate molecule for the anticonvulsant activity of P. barbatus extracts, it is possible that the bioactivity is mediated by a combination of two or more molecules. Further experiments will be required to identify the active molecules(s) and their mechanism(s) of action.

References

1. C. W. Lukhoba, M. S. J. Simmonds, and A. J. Paton, “Plectranthus: a review of ethnobotanical uses,” Journal of Ethnopharmacology, vol. 103, no. 1, pp. 1–24, 2006. View at: Publisher Site | Google Scholar
2. S. Gupta, J. N. S. Yadava, and J. S. Tandon, “Antisecretory (antidiarrhoeal) activity of Indian medicinal plants against Escherichia coli enterotoxin-induced secretion in rabbit and guinea pig ileal loop models,” International Journal of Pharmacognosy, vol. 31, no. 3, pp. 198–204, 1993. View at: Google Scholar
3. F. C. G. Almeida and I. P. Lemonica, “The toxic effects of Coleus barbatus B. on the different periods of pregnancy in rats,” Journal of Ethnopharmacology, vol. 73, no. 1-2, pp. 53–60, 2000. View at: Publisher Site | Google Scholar
4. K. Chifundera, “Contribution to the inventory of medicinal plants from the Bushi area, South Kivu Province, Democratic Republic of Congo,” Fitoterapia, vol. 72, no. 4, pp. 351–368, 2001. View at: Publisher Site | Google Scholar
5. C. C. Câmara, N. R. F. Nascimento, C. L. Macêdo-Filho, F. B. S. Almeida, and M. C. Fonteles, “Antispasmodic effect of the essential oil of Plectranthus barbatus and some major constituents on the guinea-pig ileum,” Planta Medica, vol. 69, no. 12, pp. 1080–1085, 2003. View at: Publisher Site | Google Scholar
6. F. A. Hamill, S. Apio, N. K. Mubiru et al., “Traditional herbal drugs of Southern Uganda—II: literature analysis and antimicrobial assays,” Journal of Ethnopharmacology, vol. 84, no. 1, pp. 57–78, 2003. View at: Publisher Site | Google Scholar
7. T. Johns, J. O. Kokwaro, and E. K. Kimanani, “Herbal remedies of the Luo of Siaya District, Kenya: quantitative criteria for consensus,” Economic Botany, vol. 44, pp. 369–881, 1990. View at: Google Scholar
8. A. Rajendran, N. R. Rao, K. Ravikumar, and A. N. Henry, “Some medicinal and aromatic labiates from the Peninsular India,” Journal of Non-Timber Forest Products, vol. 6, pp. 26–30, 1999. View at: Google Scholar
9. P. Cos, N. Hermans, T. De Bruyne et al., “Further evaluation of Rwandan medicinal plant extracts for their antimicrobial and antiviral activities,” Journal of Ethnopharmacology, vol. 79, no. 2, pp. 155–163, 2002. View at: Publisher Site | Google Scholar
10. B. T. Schaneberg and I. A. Khan, “Quantitative analysis of forskolin in Coleus forskohlii (Lamiaceae) by reversed-phase liquid chromatography,” Journal of AOAC International, vol. 86, no. 3, pp. 467–470, 2003. View at: Google Scholar
11. G. Philippe, L. Angenot, M. Tits, and M. Frédérich, “About the toxicity of some Strychnos species and their alkaloids,” Toxicon, vol. 44, no. 4, pp. 405–416, 2004. View at: Publisher Site | Google Scholar
12. D. B. Clifford, J. W. Olney, A. Maniotis, R. C. Collins, and C. F. Zorumski, “The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures,” Neuroscience, vol. 23, no. 3, pp. 953–968, 1987. View at: Google Scholar
13. L. J. Quintans, J. R. G. S. Almeida, J. T. Lima et al., “Plants with anticonvulsant properties—a review,” Brazilian Journal of Pharmacognosy, vol. 18, pp. 798–819, 2008. View at: Publisher Site | Google Scholar
14. H. Pérez-Saad, M. T. Buznego, M. Llanio, M. D. Fernández, and R. Menéndez, “Neuropharmacological profile of Plectranthus amboinicus (Lour.) Spreng,” Revista de Neurologia, vol. 36, no. 1, pp. 98–99, 2003. View at: Google Scholar
15. P. L. Falé, C. Borges, P. J. A. Madeira et al., “Rosmarinic acid, scutellarein $4^{\prime }$-methyl ether 7-O-glucuronide and (16S)-coleon E are the main compounds responsible for the antiacetylcholinesterase and antioxidant activity in herbal tea of Plectranthus barbatus (“falso boldo”),” Food Chemistry, vol. 114, no. 3, pp. 798–805, 2009. View at: Publisher Site | Google Scholar
16. L. J. ValdÉs, S. G. Mislankar, and A. G. Paul, “Coleus barbatus (C. forskohlii)(Lamiaceae) and the potential new drug forskolin (Coleonol),” Economic Botany, vol. 41, no. 4, pp. 474–483, 1987. View at: Publisher Site | Google Scholar
17. M. Abdel-Mogib, H. A. Albar, and S. M. Batterjee, “Chemistry of the genus Plectranthus,” Molecules, vol. 7, no. 2, pp. 271–301, 2002. View at: Google Scholar
18. R. L. De Albuquerque, M. R. Kentopff, M. I. L. Machado et al., “Abietane diterpenoids isolation from Plectranthus barbatus,” Quimica Nova, vol. 30, no. 8, pp. 1882–1886, 2007. View at: Google Scholar
19. M. Sano, A. Seto-Ohshima, and A. Mizutani, “Forskolin supresses seizures induced by pentylenetetrazol in mice,” Experientia, vol. 40, no. 11, pp. 1270–1271, 1984. View at: Google Scholar
20. M. V. Podda, E. Riccardi, M. D'Ascenzo, G. B. Azzena, and C. Grassi, “Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K⁺ currents through a cAMP-dependent protein kinase A-independent mechanism,” Neuroscience, vol. 167, no. 3, pp. 678–690, 2010. View at: Publisher Site | Google Scholar
21. M. Nakamura and IL. S. Jang, “GlyT-2 mediates the forskolin-induced increase of glycinergic transmission,” NeuroReport, vol. 167, pp. 678–690, 2010. View at: Publisher Site | Google Scholar
22. T. Mele, M. Čarman-Kržan, and D. M. Jurič, “Regulatory role of monoamine neurotransmitters in astrocytic NT-3 synthesis,” International Journal of Developmental Neuroscience, vol. 28, no. 1, pp. 13–19, 2010. View at: Publisher Site | Google Scholar
23. P. C. Maisonpierre, L. Belluscio, S. Squinto et al., “Neurotrophin-3: a neurotrophic factor related to NGF and BDNF,” Science, vol. 247, no. 4949 I, pp. 1446–1451, 1990. View at: Google Scholar
24. A. M. Davies, “Neurotrophins: neurotrophic modulation of neurite growth,” Current Biology, vol. 10, no. 5, pp. R198–R200, 2000. View at: Publisher Site | Google Scholar
25. R. R. Hicks, V. B. Martin, L. Zhang, and K. B. Seroogy, “Mild experimental brain injury differentially alters the expression of neurotrophin and neurotrophin receptor mRNAs in the hippocampus,” Experimental Neurology, vol. 160, no. 2, pp. 469–478, 1999. View at: Publisher Site | Google Scholar
26. B. Cheng and M. P. Mattson, “NT-3 and BDNF protect CNS neurons against metabolic/excitotoxic insults,” Brain Research, vol. 640, no. 1-2, pp. 56–67, 1994. View at: Google Scholar
27. S. Lee, J. Williamson, E. W. Lothman et al., “Early induction of mRNA for calbindin-D(28k) and BDNF but not NT-3 in rat hippocampus after kainic acid treatment,” Molecular Brain Research, vol. 47, no. 1-2, pp. 183–194, 1997. View at: Publisher Site | Google Scholar
28. H. Hagihara, M. Hara, K. Tsunekawa, Y. Nakagawa, M. Sawada, and K. Nakano, “Tonic-clonic seizures induce division of neuronal progenitor cells with concomitant changes in expression of neurotrophic factors in the brain of pilocarpine-treated mice,” Molecular Brain Research, vol. 139, no. 2, pp. 258–266, 2005. View at: Publisher Site | Google Scholar
29. B. Xu, B. Michalski, R. J. Racine, and M. Fahnestock, “Continuous infusion of neurotrophin-3 triggers sprouting, decreases the levels of TrkA and TrkC, and inhibits epileptogenesis and activity-dependent axonal growth in adult rats,” Neuroscience, vol. 115, no. 4, pp. 1295–1308, 2002. View at: Publisher Site | Google Scholar

Copyright

Copyright © 2012 Luciana Cristina Borges Fernandes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.