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Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats using the open field and elevated plus maze tests
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats using the open field and elevated plus maze tests Maria Thereza Gamberini a,n, Domingos Sávio Rodrigues b, Daniela Rodrigues c, Victoria Bottino Pontes d a Department of Physiological Sciences of the Santa Casa de Sao Paulo School of Medical Sciences, R. Dr. Cesário Mota Júnior, 112, Vila Buarque, São Paulo, SP, Brazil b Instituto de Botânica de São Paulo, Centro de Pesquisa Jardim Botânico e Reservas, Av. Miguel Estefano, 3031, Vila Água Funda, São Paulo, SP, Brazil c Santa Casa de Sao Paulo School of Medical Sciences, R. Dr. Cesário Mota Júnior, 61, Vila Buarque, São Paulo, SP, Brazil d A.C. Camargo Cancer Center, Rua Professor Antônio Prudente, 211, Liberdade, São Paulo, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 18 March 2015 Accepted 24 March 2015
Ethnopharmacological relevance: Pimpinella anisum L. is considered one of the first plants used for medicinal purposes. Pharmacological actions of the plant on the central nervous system have been proven but previous analyses have focused on anticonvulsant and neuroprotective actions. In traditional medicine worldwide, the use of Pimpinella is commonly recommended as a tranquilizer, although no scientific information supporting this use is available. Therefore, it was decided to investigate the central actions of the plant to observe behavioral responses, with an emphasis on the emotional component. Aim of study: To investigate the effects of the aqueous extract of Pimpinella seeds on exploratory activity and emotional behavior in rats using the open field and elevated plus maze tests. Materials and methods: Seeds of Pimpinella were extracted with distilled water, concentrated and freezedried yielding the aqueous extract(AE). Rats were divided into four groups: control(water 5 mL/kg, p.o.) and AE 0.5, 1.0 and 2.0 g/kg, p.o. Individual observations were performed in an open field and the parameters locomotor activity, rearing, grooming and defecation were recorded. In elevated plus maze test, rats were divided into four groups: control(water 5 mL/kg, p.o.) and AE 0.5, 1.0 and 2.0 g/kg, p.o. The parameters arm entries, total time spent in open and closed arms; and total number of arrivals at the end of an open or closed arm were recorded for each rat. Results: Among the parameters assessed with the open field test, only rearing was reduced in the AE 0.5 g/kg group. When AE 1.0 g/kg was administered, only the initiation of exploratory activity was delayed, without impairing the animals' general activity. The highest dose of AE (2.0 g/kg) induced a reduction in the animals' habituation during the open field test within the same session, as evidenced by the maintenance of high levels of peripheral locomotion and rearing throughout the test. On the elevated plus maze test, no alterations were observed in the responses of the animals relative to controls for all doses tested. Conclusion: These results failed to support anxiety-related central action of the aqueous extract of Pimpinella seeds, invalidating popular beliefs regarding a tranquilizing effect. However, a habituationrelated central action of the extract was demonstrated, suggesting action of bioactive compounds on central learning-related areas. The characterization of effects that may interfere with cognitive processes reinforces caution regarding indiscriminate consumption of the plant, especially in individuals with deficits, such as Alzheimer's Disease patients. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Pimpinella anisum L. Open field Elevated plus maze Rats Anxiety Habituation
n
Corresponding author. Tel.: þ 55 11 972925886; fax: þ55 11 33312008. E-mail address: [email protected] (M.T. Gamberini).
http://dx.doi.org/10.1016/j.jep.2015.03.053 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
67 68 69 70 71 72 73 74 75 76 77
M.T. Gamberini et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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1. Introduction Pimpinella anisum L.(Apiaceae) is a grassy annual herb from the Mediterranean Region and Western Asia, reaching 30–50 cm in height, with white flowers and small green to yellow seeds. Chemical analyses has revealed the presence of trans-anethole, estragole, γ-himachalene, p-anisaldehyde, and methylchavicol; as well as coumarins, scopoletin, umbelliferone, estrols, terpene hydrocarbons, polyenes and polyacetylenes as the major compounds of the plant (Gülcın et al., 2003; Shojaii and Fard, 2012). Pharmacological studies were performed on Pimpinella and various properties demonstrated such as antimicrobial, antifungal, antiviral, antioxidant, muscle relaxant, analgesic, beneficial effects on dysmenorrhea and menopausal hot flushes in women, as well as hypoglycemic and hypolipidemic effects in diabetic patients (Shojaii and Fard, 2012). Investigations of Pimpinella's actions on the nervous system have confirmed its anticonvulsant and neuroprotective effects (Pourgholami et al., 1999; Karimzadeh et al., 2012) corroborating common knowledge on its use in neurological disorders. In Brazilian traditional medicine, tea (referred to in the present study as aqueous extract) of Pimpinella prepared by infusion of the plant seeds is commonly used to induce a tranquilizing effect. Similar therapeutic effects are reported in other cultures (Atta-ur-Rahman, 2008; Gülcın et al., 2003; Tepe and Tepe, 2015), although there is no scientific information in support of this information. Therefore, the aim of the present study was to investigate possible effects of aqueous extract of Pimpinella seeds on behavioral responses in rats, with an emphasis on the emotional component. For this, open field and elevated plus maze tests, classic experimental models used to investigate the exploratory activity and emotional behavior of rodents were employed.
2. Material and methods 2.1. Animals Wistar rats (220–300 g) were kept in an environment at a controlled temperature (227 2 1C), 50% humidity level and 12 h light-dark cycle. All behavioral tests were performed between 12:00pm. and 2:00pm. All procedures were performed in strict accordance with the guidelines set by the Colégio Brasileiro de Experimentação Animal (COBEA, Brazilian Committee on Animal Research Ethics), and those of the National Institutes of Health in the ‘Guide for the Care and Use of Laboratory Animals' (NIH Publications 80-23). 2.2. Plant material The dried seeds of P. anisum L. (www.theplantlist.org, November 10, 2014) were purchased from a commercial source in São Paulo (Brazil) and kept at 8 1C. For confirmation of botanical plant identity, a sample of the seeds was grown in the Instituto Botânico de São Paulo, Secretaria do Meio Ambiente do Estado de São Paulo, Brazil. The Herbarium Register is SP 468 443. The seeds (100 g) were extracted with 1 L distilled water (70 1C, 30 min), concentrated and then freeze-dried producing aqueous extract (AE) with a 5% yield. 2.3. Bioassays 2.3.1. Open field test Rats were divided into Group 1 (control) that received water (5 mL/kg, p.o.), whereas the others three experimental groups, received AE 0.5, 1.0 and 2.0 g/kg, respectively, by gavage. Thirty
minutes after administration, the animals' spontaneous activity was evaluated in an open field test. In individual tests, the rats were placed at the same point and allowed to freely explore the apparatus for 5 min. The following measurements were then recorded: peripheral locomotor activity; central locomotor activity; number of rearings; and immobility. Grooming time and the number of defecations were also recorded (Prut and Belzung, 2003). 2.3.2. Elevated plus maze test Rats were divided into Group 1(control) that received water (5 mL/kg), whereas the others three experimental groups received AE 0.5, 1.0 and 2.0 g/kg, respectively, by gavage. Thirty minutes after administration, each rat was placed in the central square facing a closed arm and allowed to freely explore the maze for 5 min. The following measurements were recorded: arm entries; total time spent in open and closed arms; total number of arrivals at the end of an open or closed arm (Pellow et al., 1985). 2.4. Statistical analysis Results were expressed as mean 7standard error of the mean. Parameters were evaluated using analysis of variance(ANOVA), followed by Tukey's test. Differences between the two groups were assessed by Student's t-test, with a significance level of p o0.05.
3. Results In the open field test, administration of AE induced no changes in central locomotion, total time grooming or defecation when compared to controls (n ¼10) (Fig. 1A, C and F). However, initiation of exploratory activity was delayed after administration of AE 1.0 g/kg (n¼ 5) or 2.0 g/kg (n ¼5), p.o. Immobility time increased during the first (1.0 or 2.0 g/kg AE, p.o.) and second (1.0 g/kg AE, p. o.) minutes of observation relative to the control group (Fig. 1D). A reduction in exploratory activity was also observed when animals were treated with the lowest dose of the extract (0.5 g/kg AE, n¼ 5, p.o.). At this dose, rearing was reduced in the third and fourth minutes in relation to the controls (Fig. 1E). Among rats treated with the higher dose of the extract, although exploratory activity was initially reduced, levels subsequently remained elevated. Peripheral locomotion remained elevated in the third and fifth minutes relative to controls (Fig. 1B) and at the same dose, rearing was elevated in the fifth minute as compared to controls (Fig. 1E). On the elevated plus maze test, AE 0.5 g/kg (n ¼ 5), 1.0 g/kg (n ¼5) and 2.0 g/kg (n ¼5) AE, p.o. induced no changes compared to the control group (n ¼5) in any of the parameters assessed over the 5-min observation period, as shown in Fig. 2.
4. Discussion Numerous plants considered medicinal are used indiscriminately by the population, often without pharmacological validation having been performed. For such validations, specific pharmacological tests must be performed and, through objective analysis, claims confirmed or refuted. The fact that P. anisum L., is recommended in traditional medicine for its central tranquilizing action, without confirmatory scientific evidence, prompted the present study. Open field and elevated plus maze tests are classic experimental models used to investigate the exploratory activity and emotional behavior of rodents (Pellow et al., 1985; Prut and Belzung, 2003). Employing this paradigm, the behavioral response of rats treated with different doses of aqueous extract (tea) of Pimpinella
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Fig. 1. Spontaneous general activity of rats, as recorded in an open field test, after acute administration of 5 ml/kg water, p.o. (control, n¼ 10) and AE 0.5 g/kg (n ¼5), 1.0 g/kg (n¼ 5) and 2.0 g/kg (n ¼5) (p.o.). (A) Central locomotor activity; (B) peripheral locomotor activity; (C) rearing; (D) immobility; (E) grooming time; and (F) defecation, were recorded every minute during 5-minute sessions. Each point represents mean value and bar indicates S.E.M. np o 0.05 compared to control group.
was assessed. Unexpectedly, none of the doses of the aqueous extract induced changes in anxiety of the animals when evaluated on the elevated plus maze test, failing to confirm anxiolytic action of the aqueous plant extract. However, the behavioral responses of animals evaluated on the open field test were found to be modified. The most significant response was observed for the administration of the highest dose of AE. The natural reduction in exploratory activity during the open field test, as evidenced by peripheral locomotion and rearing, did not occur. Locomotion and rearing measurements are conventionally used to determine habituation learning in the open field (Thiel et al., 1998). Therefore, it was demonstrated that the administration of AE 2.0 g/kg caused a reduction in the animals' habituation to the new environment within the same session. Behavioral habituation, defined as a response decrease with repeated or continuous presentation of indifferent stimuli, is one of the most elementary forms of learning. Behavioral habituation
in rodents is analyzed in terms of exploratory behavior, especially during exposure to an open field (Leussis and Bolivar, 2006; Thiel et al., 1998). It has been shown that open field exploration and habituation are closely related to the hippocampus and its cholinergic input (Thiel et al., 1998). Behavioral activation promoted by the novel environment is positively correlated with increased hippocampal ACh levels; however, behavioral habituation to this environment is not paralleled by a decreased activation of cholinergic activity. Additionally, lesions to the hippocampus or pharmacological blockade of the cholinergic input strongly affect exploratory behavior and its habituation (Thiel et al., 1998). The close relationship between cholinergic neurotransmission and other neural pathways related to behavioral responses, such as dopaminergic neurotransmission, also involved in the process of habituation, must be considered (Gamberini et al., 2012; Leussis and Bolivar, 2006). The involvement of the cholinergic pathway has already been implicated in a pharmacological action induced by Pimpinella.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Fig. 2. Behavioral performance of rats on the elevated plus maze test after acute administration of 5 ml/kg water p.o. (control, n¼ 5) and AE 0.5 g/kg (n¼5), 1.0 g/kg (n ¼5) and 2.0 g/kg (n¼ 5) (p.o.). (A) Entries into open arms; (B) entries into closed arms; (C) time spent in open arms; (D) time spent in closed arms; (E) total number of arrivals at end of open arms, and (F) total number of arrivals at end of closed arms. Columns represent mean 7SEM.
Boskabady and Ramazani-Assari (2001) showed that the relaxant effect of aqueous and ethanolic extracts, as well as essential oil, of P. anisum on isolated guinea pig tracheal chains was attributed to inhibitory effects on muscarinic receptors. Thus, our data offer new prospects for investigating the action of bioactive compounds present in the aqueous extract of Pimpinella on central cholinergic pathways related to learning and memory. In conclusion, the results obtained in the current study add to the body of knowledge on the analysis of the central effects of Pimpinella. The importance of a critical and objective analysis in the study of medicinal plants was demonstrated. Furthermore, characterization of effects that may interfere with cognitive processes reinforces caution regarding indiscriminate consumption of the plant, especially in individuals with deficits, such as Alzheimer's Disease patients.
Acknowledgments We are grateful to Dr. Cintia Kameyama of the Núcleo de Curadoria do Herbario SP - Instituto de Botânica for providing the register of the plant and to Dr. Rosangela Simão Bianchini for botanical identification. References Atta-ur-Rahman, 2008. 1st ed.Studies in Natural Products Chemistry, 35. Elsevier Science, pp. 557–559. Boskabady, M.H., Ramazani-Assari, M., 2001. Relaxant effect of Pimpinella anisum on isolated guinea pig tracheal chains and its possible mechanism(s). J. Ethnopharmacol. 74, 83–88. Gamberini, M.T., Bolognesi, M.L., Nasello, A.G., 2012. The modulatory role of M2 muscarinic receptor on apomorphine-induced yawning and genital grooming. Neurosci. Lett. 531, 91–95.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Gülcın, İ., Oktay, M., Kıreçıc, E., Küfrevıoglu, Ö.I., 2003. Screening of antioxidant and antimicrobial activities of (Pimpinella anisum L.) seed extracts. Food Chem. 83, 371–382. Karimzadeh, F., Hosseini, M., Mangeng, D., Alavi, H., Hassanzadeh, G.R., Bayat, M., Jafarian, M., Kazemi, H., Gorji, A., 2012. Anticonvulsant and neuroprotective effects of Pimpinella anisumin rat brain. BMC Complement. Altern. Med. 12, 76. Leussis, M.P., Bolivar, V.J., 2006. Habituation in rodents: a review of behavior, neurobiology and genetics. Neurosci. Biobehav. Rev. 30, 1045–1064. Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14, 149–167. Pourgholami, M.H., Majzoob, S., Javadi, M., Kamalinejad, M., Fanaee, G.H.R., Sayyah, M., 1999. The fruit essential oil of Pimpinella anisum exerts anticonvulsant effects in mice. J. Ethnopharmacol. 66, 211–215.
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Prut, L., Belzung, C., 2003. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur. J. Pharmacol. 463, 3–33. Shojaii, A., Fard, M.A., 2012. Review of pharmacological properties and chemical constituents of Pimpinella anisum. Int. Sch. Res. Netw. Pharm. 2012, 8. http://dx. doi.org/10.5402/2012/510795, ID 510795. Tepe, A.S., Tepe, B., 2015. Tradicional use, biological activity potencial and toxicity of Pimpinella species. Ind. Crops Prod. 69, 153–166. Thiel, C.M., Huston, J.P., Schwarting, R.K., 1998. Hippocampal acetylcholine and habituation learning. Neuroscience 85, 1253–1262.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats using the open field and elevated plus maze tests Maria Thereza Gamberini a,n, Domingos Sávio Rodrigues b, Daniela Rodrigues c, Victoria Bottino Pontes d a Department of Physiological Sciences of the Santa Casa de Sao Paulo School of Medical Sciences, R. Dr. Cesário Mota Júnior, 112, Vila Buarque, São Paulo, SP, Brazil b Instituto de Botânica de São Paulo, Centro de Pesquisa Jardim Botânico e Reservas, Av. Miguel Estefano, 3031, Vila Água Funda, São Paulo, SP, Brazil c Santa Casa de Sao Paulo School of Medical Sciences, R. Dr. Cesário Mota Júnior, 61, Vila Buarque, São Paulo, SP, Brazil d A.C. Camargo Cancer Center, Rua Professor Antônio Prudente, 211, Liberdade, São Paulo, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 18 March 2015 Accepted 24 March 2015
Ethnopharmacological relevance: Pimpinella anisum L. is considered one of the first plants used for medicinal purposes. Pharmacological actions of the plant on the central nervous system have been proven but previous analyses have focused on anticonvulsant and neuroprotective actions. In traditional medicine worldwide, the use of Pimpinella is commonly recommended as a tranquilizer, although no scientific information supporting this use is available. Therefore, it was decided to investigate the central actions of the plant to observe behavioral responses, with an emphasis on the emotional component. Aim of study: To investigate the effects of the aqueous extract of Pimpinella seeds on exploratory activity and emotional behavior in rats using the open field and elevated plus maze tests. Materials and methods: Seeds of Pimpinella were extracted with distilled water, concentrated and freezedried yielding the aqueous extract(AE). Rats were divided into four groups: control(water 5 mL/kg, p.o.) and AE 0.5, 1.0 and 2.0 g/kg, p.o. Individual observations were performed in an open field and the parameters locomotor activity, rearing, grooming and defecation were recorded. In elevated plus maze test, rats were divided into four groups: control(water 5 mL/kg, p.o.) and AE 0.5, 1.0 and 2.0 g/kg, p.o. The parameters arm entries, total time spent in open and closed arms; and total number of arrivals at the end of an open or closed arm were recorded for each rat. Results: Among the parameters assessed with the open field test, only rearing was reduced in the AE 0.5 g/kg group. When AE 1.0 g/kg was administered, only the initiation of exploratory activity was delayed, without impairing the animals' general activity. The highest dose of AE (2.0 g/kg) induced a reduction in the animals' habituation during the open field test within the same session, as evidenced by the maintenance of high levels of peripheral locomotion and rearing throughout the test. On the elevated plus maze test, no alterations were observed in the responses of the animals relative to controls for all doses tested. Conclusion: These results failed to support anxiety-related central action of the aqueous extract of Pimpinella seeds, invalidating popular beliefs regarding a tranquilizing effect. However, a habituationrelated central action of the extract was demonstrated, suggesting action of bioactive compounds on central learning-related areas. The characterization of effects that may interfere with cognitive processes reinforces caution regarding indiscriminate consumption of the plant, especially in individuals with deficits, such as Alzheimer's Disease patients. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Pimpinella anisum L. Open field Elevated plus maze Rats Anxiety Habituation
n
Corresponding author. Tel.: þ 55 11 972925886; fax: þ55 11 33312008. E-mail address: [email protected] (M.T. Gamberini).
http://dx.doi.org/10.1016/j.jep.2015.03.053 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
67 68 69 70 71 72 73 74 75 76 77
M.T. Gamberini et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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1. Introduction Pimpinella anisum L.(Apiaceae) is a grassy annual herb from the Mediterranean Region and Western Asia, reaching 30–50 cm in height, with white flowers and small green to yellow seeds. Chemical analyses has revealed the presence of trans-anethole, estragole, γ-himachalene, p-anisaldehyde, and methylchavicol; as well as coumarins, scopoletin, umbelliferone, estrols, terpene hydrocarbons, polyenes and polyacetylenes as the major compounds of the plant (Gülcın et al., 2003; Shojaii and Fard, 2012). Pharmacological studies were performed on Pimpinella and various properties demonstrated such as antimicrobial, antifungal, antiviral, antioxidant, muscle relaxant, analgesic, beneficial effects on dysmenorrhea and menopausal hot flushes in women, as well as hypoglycemic and hypolipidemic effects in diabetic patients (Shojaii and Fard, 2012). Investigations of Pimpinella's actions on the nervous system have confirmed its anticonvulsant and neuroprotective effects (Pourgholami et al., 1999; Karimzadeh et al., 2012) corroborating common knowledge on its use in neurological disorders. In Brazilian traditional medicine, tea (referred to in the present study as aqueous extract) of Pimpinella prepared by infusion of the plant seeds is commonly used to induce a tranquilizing effect. Similar therapeutic effects are reported in other cultures (Atta-ur-Rahman, 2008; Gülcın et al., 2003; Tepe and Tepe, 2015), although there is no scientific information in support of this information. Therefore, the aim of the present study was to investigate possible effects of aqueous extract of Pimpinella seeds on behavioral responses in rats, with an emphasis on the emotional component. For this, open field and elevated plus maze tests, classic experimental models used to investigate the exploratory activity and emotional behavior of rodents were employed.
2. Material and methods 2.1. Animals Wistar rats (220–300 g) were kept in an environment at a controlled temperature (227 2 1C), 50% humidity level and 12 h light-dark cycle. All behavioral tests were performed between 12:00pm. and 2:00pm. All procedures were performed in strict accordance with the guidelines set by the Colégio Brasileiro de Experimentação Animal (COBEA, Brazilian Committee on Animal Research Ethics), and those of the National Institutes of Health in the ‘Guide for the Care and Use of Laboratory Animals' (NIH Publications 80-23). 2.2. Plant material The dried seeds of P. anisum L. (www.theplantlist.org, November 10, 2014) were purchased from a commercial source in São Paulo (Brazil) and kept at 8 1C. For confirmation of botanical plant identity, a sample of the seeds was grown in the Instituto Botânico de São Paulo, Secretaria do Meio Ambiente do Estado de São Paulo, Brazil. The Herbarium Register is SP 468 443. The seeds (100 g) were extracted with 1 L distilled water (70 1C, 30 min), concentrated and then freeze-dried producing aqueous extract (AE) with a 5% yield. 2.3. Bioassays 2.3.1. Open field test Rats were divided into Group 1 (control) that received water (5 mL/kg, p.o.), whereas the others three experimental groups, received AE 0.5, 1.0 and 2.0 g/kg, respectively, by gavage. Thirty
minutes after administration, the animals' spontaneous activity was evaluated in an open field test. In individual tests, the rats were placed at the same point and allowed to freely explore the apparatus for 5 min. The following measurements were then recorded: peripheral locomotor activity; central locomotor activity; number of rearings; and immobility. Grooming time and the number of defecations were also recorded (Prut and Belzung, 2003). 2.3.2. Elevated plus maze test Rats were divided into Group 1(control) that received water (5 mL/kg), whereas the others three experimental groups received AE 0.5, 1.0 and 2.0 g/kg, respectively, by gavage. Thirty minutes after administration, each rat was placed in the central square facing a closed arm and allowed to freely explore the maze for 5 min. The following measurements were recorded: arm entries; total time spent in open and closed arms; total number of arrivals at the end of an open or closed arm (Pellow et al., 1985). 2.4. Statistical analysis Results were expressed as mean 7standard error of the mean. Parameters were evaluated using analysis of variance(ANOVA), followed by Tukey's test. Differences between the two groups were assessed by Student's t-test, with a significance level of p o0.05.
3. Results In the open field test, administration of AE induced no changes in central locomotion, total time grooming or defecation when compared to controls (n ¼10) (Fig. 1A, C and F). However, initiation of exploratory activity was delayed after administration of AE 1.0 g/kg (n¼ 5) or 2.0 g/kg (n ¼5), p.o. Immobility time increased during the first (1.0 or 2.0 g/kg AE, p.o.) and second (1.0 g/kg AE, p. o.) minutes of observation relative to the control group (Fig. 1D). A reduction in exploratory activity was also observed when animals were treated with the lowest dose of the extract (0.5 g/kg AE, n¼ 5, p.o.). At this dose, rearing was reduced in the third and fourth minutes in relation to the controls (Fig. 1E). Among rats treated with the higher dose of the extract, although exploratory activity was initially reduced, levels subsequently remained elevated. Peripheral locomotion remained elevated in the third and fifth minutes relative to controls (Fig. 1B) and at the same dose, rearing was elevated in the fifth minute as compared to controls (Fig. 1E). On the elevated plus maze test, AE 0.5 g/kg (n ¼ 5), 1.0 g/kg (n ¼5) and 2.0 g/kg (n ¼5) AE, p.o. induced no changes compared to the control group (n ¼5) in any of the parameters assessed over the 5-min observation period, as shown in Fig. 2.
4. Discussion Numerous plants considered medicinal are used indiscriminately by the population, often without pharmacological validation having been performed. For such validations, specific pharmacological tests must be performed and, through objective analysis, claims confirmed or refuted. The fact that P. anisum L., is recommended in traditional medicine for its central tranquilizing action, without confirmatory scientific evidence, prompted the present study. Open field and elevated plus maze tests are classic experimental models used to investigate the exploratory activity and emotional behavior of rodents (Pellow et al., 1985; Prut and Belzung, 2003). Employing this paradigm, the behavioral response of rats treated with different doses of aqueous extract (tea) of Pimpinella
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Fig. 1. Spontaneous general activity of rats, as recorded in an open field test, after acute administration of 5 ml/kg water, p.o. (control, n¼ 10) and AE 0.5 g/kg (n ¼5), 1.0 g/kg (n¼ 5) and 2.0 g/kg (n ¼5) (p.o.). (A) Central locomotor activity; (B) peripheral locomotor activity; (C) rearing; (D) immobility; (E) grooming time; and (F) defecation, were recorded every minute during 5-minute sessions. Each point represents mean value and bar indicates S.E.M. np o 0.05 compared to control group.
was assessed. Unexpectedly, none of the doses of the aqueous extract induced changes in anxiety of the animals when evaluated on the elevated plus maze test, failing to confirm anxiolytic action of the aqueous plant extract. However, the behavioral responses of animals evaluated on the open field test were found to be modified. The most significant response was observed for the administration of the highest dose of AE. The natural reduction in exploratory activity during the open field test, as evidenced by peripheral locomotion and rearing, did not occur. Locomotion and rearing measurements are conventionally used to determine habituation learning in the open field (Thiel et al., 1998). Therefore, it was demonstrated that the administration of AE 2.0 g/kg caused a reduction in the animals' habituation to the new environment within the same session. Behavioral habituation, defined as a response decrease with repeated or continuous presentation of indifferent stimuli, is one of the most elementary forms of learning. Behavioral habituation
in rodents is analyzed in terms of exploratory behavior, especially during exposure to an open field (Leussis and Bolivar, 2006; Thiel et al., 1998). It has been shown that open field exploration and habituation are closely related to the hippocampus and its cholinergic input (Thiel et al., 1998). Behavioral activation promoted by the novel environment is positively correlated with increased hippocampal ACh levels; however, behavioral habituation to this environment is not paralleled by a decreased activation of cholinergic activity. Additionally, lesions to the hippocampus or pharmacological blockade of the cholinergic input strongly affect exploratory behavior and its habituation (Thiel et al., 1998). The close relationship between cholinergic neurotransmission and other neural pathways related to behavioral responses, such as dopaminergic neurotransmission, also involved in the process of habituation, must be considered (Gamberini et al., 2012; Leussis and Bolivar, 2006). The involvement of the cholinergic pathway has already been implicated in a pharmacological action induced by Pimpinella.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Fig. 2. Behavioral performance of rats on the elevated plus maze test after acute administration of 5 ml/kg water p.o. (control, n¼ 5) and AE 0.5 g/kg (n¼5), 1.0 g/kg (n ¼5) and 2.0 g/kg (n¼ 5) (p.o.). (A) Entries into open arms; (B) entries into closed arms; (C) time spent in open arms; (D) time spent in closed arms; (E) total number of arrivals at end of open arms, and (F) total number of arrivals at end of closed arms. Columns represent mean 7SEM.
Boskabady and Ramazani-Assari (2001) showed that the relaxant effect of aqueous and ethanolic extracts, as well as essential oil, of P. anisum on isolated guinea pig tracheal chains was attributed to inhibitory effects on muscarinic receptors. Thus, our data offer new prospects for investigating the action of bioactive compounds present in the aqueous extract of Pimpinella on central cholinergic pathways related to learning and memory. In conclusion, the results obtained in the current study add to the body of knowledge on the analysis of the central effects of Pimpinella. The importance of a critical and objective analysis in the study of medicinal plants was demonstrated. Furthermore, characterization of effects that may interfere with cognitive processes reinforces caution regarding indiscriminate consumption of the plant, especially in individuals with deficits, such as Alzheimer's Disease patients.
Acknowledgments We are grateful to Dr. Cintia Kameyama of the Núcleo de Curadoria do Herbario SP - Instituto de Botânica for providing the register of the plant and to Dr. Rosangela Simão Bianchini for botanical identification. References Atta-ur-Rahman, 2008. 1st ed.Studies in Natural Products Chemistry, 35. Elsevier Science, pp. 557–559. Boskabady, M.H., Ramazani-Assari, M., 2001. Relaxant effect of Pimpinella anisum on isolated guinea pig tracheal chains and its possible mechanism(s). J. Ethnopharmacol. 74, 83–88. Gamberini, M.T., Bolognesi, M.L., Nasello, A.G., 2012. The modulatory role of M2 muscarinic receptor on apomorphine-induced yawning and genital grooming. Neurosci. Lett. 531, 91–95.
Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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Please cite this article as: Gamberini, M.T., et al., Effects of the aqueous extract of Pimpinella anisum L. seeds on exploratory activity and emotional behavior in rats.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.053i
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