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Hydroalcoholic extract and fractions of Davilla rugosa Poiret: effects on spontaneous motor activity and elevated plus-maze behavior
Journal of Ethnopharmacology 72 (2000) 61 – 67 www.elsevier.com/locate/jethpharm
Hydroalcoholic extract and fractions of Da6illa rugosa Poiret: effects on spontaneous motor activity and elevated plus-maze behavior L. Guaraldo *, D.A. Chagas, A.C. Konno, G.P. Korn, T. Pfiffer, A.G. Nasello Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Cieˆncias Me´dicas da Santa Casa de Sa˜o Paulo, Rua Dr. Cesa´rio Motta Jr., 61 Santa Cecı´lia, 01277 -900 Sa˜o Paulo, SP, Brazil Received 11 June 1999; received in revised form 7 February 2000; accepted 22 February 2000
Abstract Da6illa rugosa Poiret is commonly used in Brazilian folk medicine. The use as stimulant induced us to study the effects on motor activity and anxiety using an open-field and an elevated plus-maze, respectively. The hydroalcoholic extract of the stems (HE) was fractionated with chloroform (CF), chloroform/ethyl acetate (CAF), ethyl acetate (AF), ethyl acetate/ethanol (AEF), ethanol (EF) and ethanol/water (EWF). Rats were treated orally with HE (7.5, 15, 30 or 60 mg/kg) or fractions (15 mg/kg). In the open-field, HE (15 mg/kg), AEF, EF and EWF increased locomotion frequency and decreased immobility time; the contrary was observed with 30 and 60 mg/kg of HE. These doses also increased defecation. No effects were observed with 7.5 mg/kg of HE, CF, CAF or AF, except for an increase in defecation induced by AF. In the elevated plus-maze, total entries and number of entries into the open and closed arms and the time spent in the open arms and its percentage were increased only with 15 mg/kg of HE. The open-field results suggest that the drug increases motor activity (stimulant effect) and that the active components are in the three more polar fractions. An anxiolytic effect was observed only with the HE. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Da6illa rugosa Poiret; Dilleniaceae; Stimulant; Anxiolytic, CNS activity
1. Introduction Da6illa rugosa Poiret, Dilleniaceae, is a liana known commonly as Cipo´-Caboclo, which * Corresponding author. Fax: +55-11-2202008. E-mail address: [email protected] (L. Guaraldo).
grows in Brazil and in other countries of Central and South America. Since it was discovered by the Carijo´ Indians, its stems are widely used for the preparation of anti-inflammatory, antiulcer, purgative, stimulant, aphrodisiac and tonic hydroalcoholic extracts and aqueous infusions (Le Cointe, 1934; Silva, 1935; Correˆa, 1984).
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 9 8 - 7
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L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
In previous chemical studies it was shown that the main constituents of the stems of D. rugosa Poiret are flavonoids, saponins and mucilage (Matheucci, 1996). Flavonoids such myricetin, quercetin, myricetin – 3-rhamnoside, quercetin – 3rhamnoside and kaempferol were isolated from the leaves (Gurni and Kubtzki, 1981), and the alkaloid caffeine was isolated from the seeds (Freise, 1935). Pharmacological investigations carried out with hydroalcoholic extracts of the stems from this species have demonstrated a gastric antiulcer activity in many experimental models in rats (Matheucci, 1996), and an anti-inflammatory effect on acute inflammatory processes (Abel et al., 1998). Although preliminary chemical and pharmacological studies with D. rugosa have been undertaken, there are not data about the pharmacological effects of this species on behavior and central nervous system (CNS). The aim of the present study was to evaluate the effects of the hydroalcoholic extract and its fractions on the spontaneous motor activity and plus-maze behavior of rats.
2. Methodology
acetate fraction, CAF), ethyl acetate (ethyl acetate fraction, AF), ethyl acetate/ethanol — 1: 1 (ethyl acetate/ethanol fraction, AEF), ethanol (ethanol fraction, EF) and ethanol/water — 1: 1 (ethanol/ water fraction, EWF), under shaking for 30 min. The fractions were dried under vacuum and freeze-dried and were equivalent to 2.32, 0.84, 0.35, 3.12, 3.14 and 83.80% of the dry HE, respectively.
2.2. Animals A total of 273 male adults rats from our colony (Wistar origin) weighing 200–250 g were used in these experiments. Naive rats were used in the open field test and the same ones were utilized in the plus-maze test. The animals were housed in polypropylene cages (32×40×15 cm3), six per cage, in controlled temperature (22–23°C) and artificially lighted (12 h light and 12 h dark, light on at 06:00 h) rooms with food and water ad libitum. On the day of the experiment, the rats were food deprived at 06:00 h and placed in the experimental room at 08:00 h. All experiments started at 12:00 h. The animals were treated with HE (7.5, 15, 30 and 60 mg/kg, p.o.) or the fractions (15 mg/kg, p.o.) 30 min before the tests. Control group received water. Days of experiments and extracts were randomized in all groups.
2.1. Plant material and extraction 2.3. Open-field beha6ior The stems of D. rugosa Poiret (Herbarium number SPF-103711) were collected in the State of Sa˜o Paulo, Brazil, and identified at the Botanic Institute, University of Sa˜o Paulo. The plant material was dried at 40°C with air circulation, ground, and extracted with 70% ethanol by percolation at room temperature. The extracts were dried at 40°C under vacuum and finally freezedried (Farmacope´ia dos Estados Unidos do Brasil, 1959). The pharmacological assays were carried out with aqueous suspensions of the dried extract (HE). The doses are expressed as mg of dried extract/kg per rat (1.0 g of dry extract is equivalent to 8.13 g dried stem). The HE was fractionated by increasing polarity with chloroform (chloroform fraction, CF), chloroform/ethyl acetate — 1:1 (chloroform/ethyl
Spontaneous motor activity was monitored for 5 min in an open-field as described previously (Broadhurst, 1960; Felicio et al., 1987; Nasello et al., 1998). Briefly, the structure consisted of a circular arena composed of a hardboard floor (diameter 99 cm) with a surrounding wall (30 cm height), both made of white painted wood. The floor was divided into 46 parts using circles and radial segments. The apparatus was placed in a sound-attenuated room, 48 cm above the floor. At the start of testing the animals were individually placed in the center of the arena and the following parameters were recorded: total locomotion (number of floor units entered), percentage of central locomotion, rearing frequency (number of times the animal stood on its hind legs), defeca-
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
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tion units (number of boli), immobility time, and grooming time. Before introducing each animal, the arena was washed with 5% alcohol to eliminate the possible bias due the odor left by the previous subjects.
Fig. 2. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 7.5, 15, 30 and 60 mg/kg, p.o.) and water (control) on percentage of central locomotion (A), grooming time (B) and defecation (C) in rats, in the open-field test. Data are means 9S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the respective controls (* PB 0.05 — ANOVA/Tukey– Kramer test).
2.4. Ele6ated plus-maze (EPM) beha6ior
Fig. 1. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 7.5, 15, 30 and 60 mg/kg, p.o.) and water (control) on total locomotion (A), rearing (B) and immobility time (C) in rats, in the open-field test. Data are means 9S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the respective controls (* PB 0.05 — ANOVA/Tukey– Kramer test).
Immediately after the open-field test, a 5-min elevated plus-maze test was performed. The apparatus consisted of a wood plus-maze elevated 50 cm from the floor and comprising two opposite open arms, 50× 10 cm, crossed at right angles by two arms of the same dimensions enclosed by 40 cm high walls, having an open roof (Zangrossi et
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
64
al., 1992; Silva et al., 1995, 1997; Nasello et al., 1998). In order to avoid falls, the open arms were surrounded by a 0.5-cm high edge. The maze was placed inside a sound-attenuated room. Subjects were placed initially in the center plataform of the maze facing an open arm. The behaviors recorded were: number of total entries into either arm, number of entries into open and closed arms, percentage of entries into open arms, time spent in the open arms, and percentage of time spent in the open arms. The test apparatus was thoroughly cleaned between subjects.
decreased total locomotion and increased immobility time. The dose of 7.5 mg/kg was ineffective. Rearing and grooming were not modified by any dose used. Defecation was increased with the doses of 30 and 60 mg/kg. The same dose (15 mg/kg) was used for all HE fractions and the results are shown in Table 1. EWF, EF and AEF increased total locomotion and decreased the duration of immobility. AEF also increased rearing. The others parameters were not affected. No effects were observed with AF, CAF and CF, except for an increase in defecation with AF treatment.
2.5. Statistical analysis 3.2. EPM beha6ior Open-field parameters were compared using ANOVA followed by the Tukey – Kramer test. Results obtained in the EPM were tested using Kruskall–Wallis ANOVA followed by the Mann–Whitney U test. In all cases differences were considered significant if P B 0.05.
The dose of 15 mg/kg of HE increased total entries, open and closed arm entries and time and percentage of time spent in the open arm. No other effects were observed with the other doses of HE or with any fraction (Fig. 3 and Table 2).
3. Results
4. Discussion and conclusion
3.1. Open-field beha6ior
The present results show that the HE of D. rugosa Poiret modifies general activity in a dosedependent manner. The lower dose (7.5 mg/kg) was ineffective; 15 mg/kg increased general activity as expressed by higher ambulation and lower immobility time. Both 30 and 60 mg/kg decreased general activity as shown by decreased ambula-
The results obtained following oral administration of HE are summarized in Figs. 1 and 2. The dose of 15 mg/kg increased total locomotion and decreased the duration of immobility. Doses of 30 and 60 mg/kg had the opposite effects, i.e. they
Table 1 Effect of CF, CAF, AF, AEF, EF and EWF of the D. rugosa Poiret hydroalcoholic extract on open-field behavior in ratsa Treatment
Total locomotion
Rearing
Immobility time
% Central locomotion
Grooming time
Defecation
Control (12) CF (12) CAF (12) Control (14) AF (13) AEF (13) Control (51) EF (26) EWF (27)
107.8 9 12.5 118.1 9 9.4 115.1 99.1 116.9 910.0 118.5 99.4 150.9 911.4* 97.7 93.4 108.9 93.4* 112.7 96.0*
28.39 2.0 30.29 3.6 24.39 3.0 24.79 3.1 29.89 2.5 33.69 2.1* 21.9 9 1.1 25.39 1.6 23.8 9 2.4
17.79 6.3 9.6 92.3 19.295.8 21.69 7.3 9.49 2.0 4.59 1.0* 28.99 3.4 10.992.7* 14.7 92.7*
8.1 9 1.9 7.3 9 1.4 6.4 90.8 8.2 91.7 7.3 9 1.2 8.39 1.0 7.9 9 0.6 8.5 9 1.2 7.8 9 0.8
43.1 9 7.6 38.49 7.5 33.9 9 8.1 33.9 9 6.6 41.79 8.8 26.59 8.0 42.59 4.2 35.9 9 3.8 40.29 5.4
2.7 91.7 2.9 90.6 2.7 90.4 3.3 90.7 6.1 90.7* 4.2 90.8 2.6 90.3 2.8 90.4 2.6 90.4
Data are means 9S.E.M.; the number of animals is given in parenthesis. * PB0.05 — ANOVA/Tukey–Kramer test, different from the respective controls.
a
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
Fig. 3. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 15 and 30 mg/kg, p.o.) and water (control) on entries into open (OAE) and closed arms (CAE) and total entries (TE) (A); time in the open (TOA) and closed arms (TCA) (B);% OAE and TOA (C) and elevated plus-maze behavior in rats. Data are means9 S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the control (* PB 0.05, ** P B 0.01 — Kruskall–Wallis/Mann–Whitney’s U test).
tion and increased immobility time. The effects of these last two doses were not different, suggesting that 30 mg/kg have the maximum effect. There were no modifications in any of the other parame-
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ters assessed in the open-field at dose used, except for an increase in defecation observed with the dose of 60 mg/kg. The results with 15 mg/kg agree with the popular use of the plant as a stimulant (1–3). Concerning the fractions, AEF, EF and EWF shared the stimulant effect with 15 mg/kg of HE, observed on the same parameters of the openfield. An additional increase in rearing was observed only with AEF. The only effect of AF was an increase in defecation. The effect of HE 30 and 60 mg/kg and AF on defecation suggests an action on intestinal activity and supports the popular use of the plant as purgative (Le Cointe, 1934; Silva, 1935; Correˆa, 1984). The presence of saponins in the HE (Matheucci, 1996) may be related to the depressing effects on general activity. It has been described that some saponins decrease general activity by modifying dopamine receptors (Kim et al., 1995a,b, 1998). On the other hand, concerning the hyperactivity observed with HE 15 mg/kg, AEF, EF and EWF may assume that: (a) the mechanisms involved are not dopaminergic; (b) the saponins present are not those which modify the dopaminergic responses; or (c) the quantities of these substances in the extract and fractions are not sufficient to elicit the effects described. Indeed other neurotransmitters may be involved in present effects, we did not find any other relations between the chemical components of the plant and neurotransmitters, except for GABA and flavonoids (see below). Albeit two doses of HE (15 and 30 mg/kg) and all the fractions were tested in the EPM, only 15 mg/kg of HE was effective. An increase in number of total entries and a higher percentage of time spent in the open arm was recorded. This higher number of total entries agrees with the increase in locomotion observed in the open-field test. Longer time spent into the open arms (together with the higher percentage of central locomotion recorded in the open-field test) suggests an anxiolytic effect (Zangrossi et al., 1992; Silva et al., 1995, 1997; Nasello et al., 1998). This is the same dose that has been reported to protect from stressinduced gastric lesions (Matheucci, 1996). Flavonoids are present in the HE and several lines
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Table 2 Effect of CF, CAF, AF, AEF, EF and EWF of the D. rugosa Poiret hydroalcoholic extract on elevated plus-maze behavior in ratsa Treatment
Total entries
Open arm entries
% Open arm entries Time in open arms
% Time in open arms
Control (12) CF (12) CAF (12) Control (14) AF (13) AEF (13) Control (45) EF (29) EWF (29)
7.5 91.4 8.891.2 9.7 91.4 7.0 91.1 6.4 90.9 9.6 91.1 5.5 90.5 6.0 90.8 6.8 90.8
3.99 1.0 4.09 0.8 4.49 0.9 2.79 0.7 2.39 0.7 4.19 0.9 2.39 0.3 2.99 0.5 3.49 0.5
37.4 98.1 43.3 9 3.5 45.8 96.5 30.1 95.7 26.4 97.1 38.1 96.4 33.3 94.1 43.6 95.0 43.9 94.9
17.8 95.5 16.6 93.6 23.1 97.9 10.5 93.0 6.5 92.9 15.5 93.8 13.3 93.1 18.7 94.5 18.6 94.1
a
53.4 916.5 49.9 910.8 69.3 9 23.8 31.4 99.1 21.2 9 8.5 50.0 9 11.5 39.8 99.4 56.0 9 13.5 55.7 9 12.3
Data are means 9S.E.M.; the number of animals is given in parenthesis.
of evidence show that natural and sinthetic flavonoids are potent anxiolytic agents without sedative, myorelaxant or amnestic effects. It is known the participation of GABA in these effects. (Haberlein et al., 1994; Marder et al., 1996; Medina et al., 1997). In conclusion, the present study showed that the HE of D. rugosa Poiret has a biphasic effect on general activity according to the dose used. The active compounds for the stimulant effects are in the three more polar fractions. Interestingly, the same dose that induces higher motor activity is anxiolytic. HE 30 and 60 mg/kg and AF may be also effective on intestinal activity. However, further studies including toxicologic evaluations and the use of more purified fractions are necessary to confirm and extend the present findings.
References Abel, M.N.C., Cury, A.N., Figueiredo, C.C., Matheucci, L.G., Nasello, A.G., 1998. Effects of Da6illa rugosa Poiret hydroalcoholic extract on carrageenin induced inflammation in rats. Naunyn-Schmiedeberg’s Archives of Pharmacology 358 (Suppl. 2), R494. Broadhurst, P.L., 1960. Experiments in psychogenetics. In: Eisenk, H.J. (Ed.), Experiments in Personality. Routlege and Kevan Paul, London. Correˆa, M.P., 1984. Diciona´rio das plantas u´teis do Brasil e das exo´ticas cultivadas. Imprensa Nacional, Rio de Janeiro, vol. 3, pp. 266–267. Farmacope´ia dos Estados Unidos do Brasil, 1959. Siqueira, Sa˜o Paulo, 2 edn., pp. 448–449.
Felicio, L.F., Nasello, A.G., Palermo-Neto, J., 1987. Dopaminergic supersensitivity after long-term bromopride treatment. Physiology and Behavior 4, 433 – 437. Freise, F.W., 1935. Das Vorkommen von Koffein in brasilianischen Heilplanzen. Pharmazeutische Zentralhalle fur Deutschland 76, 704 – 706. Gurni, A.A., Kubtzki, K., 1981. Flavonoid chemistry and systematics of the Dilleniaceae. Biochemical Systematics and Ecology 9 (2/3), 109 – 114. Haberlein, H., Tschiersch, K.P., Schafer, H.L., 1994. Flavonoids from Leptospermum scoparium with affinity to the benzodiazepine receptor characterized by structure activity relationships and in vivo studies of a plant extract. Pharmazie 49 (12), 912 – 922. Kim, H.S., Kang, J.G., Rheu, H.M., Cho, D.H., Oh, K.W., 1995. Blockade by ginseng total saponin of the development of methamphetamine reverse tolerance and dopamine receptor supersensitivity in mice. Planta Medica 61 (1), 22 – 25. Kim, H.S., Kang, J.G., Seong, Y.H., Nam, K.Y., Oh, K.W., 1995. Blockade by ginseng total saponin of the development of cocaine induced reverse tolerance and dopamine receptor supersensitivity in mice. Pharmacology, Biochemistry and Behavior 50 (1), 23 – 27. Kim, H.S., Jang, C.G., Oh, K.W., Oh, S., Rheu, H.M., Rhee, G.S., Seong, Y.H., Park, W.K., 1998. Effects of ginseng total saponin on morphine-induced hyperactivity and conditioned place preference in mice. Journal of Ethnopharmacology 60 (1), 33 – 42. Le Cointe, P., 1934. A amazoˆnia brasileira III. Livraria Cla´ssica, Bele´m, p. 123. Marder, M., Viola, H., Wasowski, C., Wolfman, C., Waterman, P.G., Cassels, B.K., Medina, J.G., Paladini, A.C., 1996. 6-Bromoflavone, a high affinity ligand for the central benzodiazepine receptors in a member of family of active flavonoids. Biochemical and Biophysical Research Communications 223 (2), 384 – 389. Matheucci, L.G., 1996. Master Thesis, Universidade de Sa˜o Paulo, Brazil.
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67 Medina, J.H., Viola, H., Wolfman, C., Marder, M., Wasowski, C., Calvo, D., Paladini, A.C., 1997. Overview — flavonoids: a new family of benzodiazepine receptor ligands. Neurochemical Research 22 (4), 419–425. Nasello, A.G., Machado, C., Bastos, J.F., Felicio, L.F., 1998. Sudden darkness induces a high activity-low anxiety state in male and female rats. Physiology and Behavior 63 (3), 451 – 454. Silva, R.A.D., 1935. Plantas medicinais do Brasil — ‘Cipo´ Caboclo’. Revista da Flora Medicinal 2 (2), 69–78.
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Silva, M.R.P., Felicio, L.F., Nasello, A.G., Bernardi, M.M., 1995. Is perinatal picrotoxin anxiogenic? Brazilian Journal of Medical and Biological Research 28, 663 – 666. Silva, M.R.P., Bernardi, M.M., Nasello, A.G., Felicio, L.F., 1997. Infuence of lactation on motor activity and elevated plus maze behavior. Brazilian Journal of Medical and Biological Research 30, 241 – 244. Zangrossi, H., Leite, J.R., Graeff, F.G., 1992. Anxiolytic effect of carbamazepine in the elevated plus-maze: possible role of adenosine. Psychopharmacology 100, 85 – 89.
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Hydroalcoholic extract and fractions of Da6illa rugosa Poiret: effects on spontaneous motor activity and elevated plus-maze behavior L. Guaraldo *, D.A. Chagas, A.C. Konno, G.P. Korn, T. Pfiffer, A.G. Nasello Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Cieˆncias Me´dicas da Santa Casa de Sa˜o Paulo, Rua Dr. Cesa´rio Motta Jr., 61 Santa Cecı´lia, 01277 -900 Sa˜o Paulo, SP, Brazil Received 11 June 1999; received in revised form 7 February 2000; accepted 22 February 2000
Abstract Da6illa rugosa Poiret is commonly used in Brazilian folk medicine. The use as stimulant induced us to study the effects on motor activity and anxiety using an open-field and an elevated plus-maze, respectively. The hydroalcoholic extract of the stems (HE) was fractionated with chloroform (CF), chloroform/ethyl acetate (CAF), ethyl acetate (AF), ethyl acetate/ethanol (AEF), ethanol (EF) and ethanol/water (EWF). Rats were treated orally with HE (7.5, 15, 30 or 60 mg/kg) or fractions (15 mg/kg). In the open-field, HE (15 mg/kg), AEF, EF and EWF increased locomotion frequency and decreased immobility time; the contrary was observed with 30 and 60 mg/kg of HE. These doses also increased defecation. No effects were observed with 7.5 mg/kg of HE, CF, CAF or AF, except for an increase in defecation induced by AF. In the elevated plus-maze, total entries and number of entries into the open and closed arms and the time spent in the open arms and its percentage were increased only with 15 mg/kg of HE. The open-field results suggest that the drug increases motor activity (stimulant effect) and that the active components are in the three more polar fractions. An anxiolytic effect was observed only with the HE. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Da6illa rugosa Poiret; Dilleniaceae; Stimulant; Anxiolytic, CNS activity
1. Introduction Da6illa rugosa Poiret, Dilleniaceae, is a liana known commonly as Cipo´-Caboclo, which * Corresponding author. Fax: +55-11-2202008. E-mail address: [email protected] (L. Guaraldo).
grows in Brazil and in other countries of Central and South America. Since it was discovered by the Carijo´ Indians, its stems are widely used for the preparation of anti-inflammatory, antiulcer, purgative, stimulant, aphrodisiac and tonic hydroalcoholic extracts and aqueous infusions (Le Cointe, 1934; Silva, 1935; Correˆa, 1984).
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 9 8 - 7
62
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
In previous chemical studies it was shown that the main constituents of the stems of D. rugosa Poiret are flavonoids, saponins and mucilage (Matheucci, 1996). Flavonoids such myricetin, quercetin, myricetin – 3-rhamnoside, quercetin – 3rhamnoside and kaempferol were isolated from the leaves (Gurni and Kubtzki, 1981), and the alkaloid caffeine was isolated from the seeds (Freise, 1935). Pharmacological investigations carried out with hydroalcoholic extracts of the stems from this species have demonstrated a gastric antiulcer activity in many experimental models in rats (Matheucci, 1996), and an anti-inflammatory effect on acute inflammatory processes (Abel et al., 1998). Although preliminary chemical and pharmacological studies with D. rugosa have been undertaken, there are not data about the pharmacological effects of this species on behavior and central nervous system (CNS). The aim of the present study was to evaluate the effects of the hydroalcoholic extract and its fractions on the spontaneous motor activity and plus-maze behavior of rats.
2. Methodology
acetate fraction, CAF), ethyl acetate (ethyl acetate fraction, AF), ethyl acetate/ethanol — 1: 1 (ethyl acetate/ethanol fraction, AEF), ethanol (ethanol fraction, EF) and ethanol/water — 1: 1 (ethanol/ water fraction, EWF), under shaking for 30 min. The fractions were dried under vacuum and freeze-dried and were equivalent to 2.32, 0.84, 0.35, 3.12, 3.14 and 83.80% of the dry HE, respectively.
2.2. Animals A total of 273 male adults rats from our colony (Wistar origin) weighing 200–250 g were used in these experiments. Naive rats were used in the open field test and the same ones were utilized in the plus-maze test. The animals were housed in polypropylene cages (32×40×15 cm3), six per cage, in controlled temperature (22–23°C) and artificially lighted (12 h light and 12 h dark, light on at 06:00 h) rooms with food and water ad libitum. On the day of the experiment, the rats were food deprived at 06:00 h and placed in the experimental room at 08:00 h. All experiments started at 12:00 h. The animals were treated with HE (7.5, 15, 30 and 60 mg/kg, p.o.) or the fractions (15 mg/kg, p.o.) 30 min before the tests. Control group received water. Days of experiments and extracts were randomized in all groups.
2.1. Plant material and extraction 2.3. Open-field beha6ior The stems of D. rugosa Poiret (Herbarium number SPF-103711) were collected in the State of Sa˜o Paulo, Brazil, and identified at the Botanic Institute, University of Sa˜o Paulo. The plant material was dried at 40°C with air circulation, ground, and extracted with 70% ethanol by percolation at room temperature. The extracts were dried at 40°C under vacuum and finally freezedried (Farmacope´ia dos Estados Unidos do Brasil, 1959). The pharmacological assays were carried out with aqueous suspensions of the dried extract (HE). The doses are expressed as mg of dried extract/kg per rat (1.0 g of dry extract is equivalent to 8.13 g dried stem). The HE was fractionated by increasing polarity with chloroform (chloroform fraction, CF), chloroform/ethyl acetate — 1:1 (chloroform/ethyl
Spontaneous motor activity was monitored for 5 min in an open-field as described previously (Broadhurst, 1960; Felicio et al., 1987; Nasello et al., 1998). Briefly, the structure consisted of a circular arena composed of a hardboard floor (diameter 99 cm) with a surrounding wall (30 cm height), both made of white painted wood. The floor was divided into 46 parts using circles and radial segments. The apparatus was placed in a sound-attenuated room, 48 cm above the floor. At the start of testing the animals were individually placed in the center of the arena and the following parameters were recorded: total locomotion (number of floor units entered), percentage of central locomotion, rearing frequency (number of times the animal stood on its hind legs), defeca-
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
63
tion units (number of boli), immobility time, and grooming time. Before introducing each animal, the arena was washed with 5% alcohol to eliminate the possible bias due the odor left by the previous subjects.
Fig. 2. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 7.5, 15, 30 and 60 mg/kg, p.o.) and water (control) on percentage of central locomotion (A), grooming time (B) and defecation (C) in rats, in the open-field test. Data are means 9S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the respective controls (* PB 0.05 — ANOVA/Tukey– Kramer test).
2.4. Ele6ated plus-maze (EPM) beha6ior
Fig. 1. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 7.5, 15, 30 and 60 mg/kg, p.o.) and water (control) on total locomotion (A), rearing (B) and immobility time (C) in rats, in the open-field test. Data are means 9S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the respective controls (* PB 0.05 — ANOVA/Tukey– Kramer test).
Immediately after the open-field test, a 5-min elevated plus-maze test was performed. The apparatus consisted of a wood plus-maze elevated 50 cm from the floor and comprising two opposite open arms, 50× 10 cm, crossed at right angles by two arms of the same dimensions enclosed by 40 cm high walls, having an open roof (Zangrossi et
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
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al., 1992; Silva et al., 1995, 1997; Nasello et al., 1998). In order to avoid falls, the open arms were surrounded by a 0.5-cm high edge. The maze was placed inside a sound-attenuated room. Subjects were placed initially in the center plataform of the maze facing an open arm. The behaviors recorded were: number of total entries into either arm, number of entries into open and closed arms, percentage of entries into open arms, time spent in the open arms, and percentage of time spent in the open arms. The test apparatus was thoroughly cleaned between subjects.
decreased total locomotion and increased immobility time. The dose of 7.5 mg/kg was ineffective. Rearing and grooming were not modified by any dose used. Defecation was increased with the doses of 30 and 60 mg/kg. The same dose (15 mg/kg) was used for all HE fractions and the results are shown in Table 1. EWF, EF and AEF increased total locomotion and decreased the duration of immobility. AEF also increased rearing. The others parameters were not affected. No effects were observed with AF, CAF and CF, except for an increase in defecation with AF treatment.
2.5. Statistical analysis 3.2. EPM beha6ior Open-field parameters were compared using ANOVA followed by the Tukey – Kramer test. Results obtained in the EPM were tested using Kruskall–Wallis ANOVA followed by the Mann–Whitney U test. In all cases differences were considered significant if P B 0.05.
The dose of 15 mg/kg of HE increased total entries, open and closed arm entries and time and percentage of time spent in the open arm. No other effects were observed with the other doses of HE or with any fraction (Fig. 3 and Table 2).
3. Results
4. Discussion and conclusion
3.1. Open-field beha6ior
The present results show that the HE of D. rugosa Poiret modifies general activity in a dosedependent manner. The lower dose (7.5 mg/kg) was ineffective; 15 mg/kg increased general activity as expressed by higher ambulation and lower immobility time. Both 30 and 60 mg/kg decreased general activity as shown by decreased ambula-
The results obtained following oral administration of HE are summarized in Figs. 1 and 2. The dose of 15 mg/kg increased total locomotion and decreased the duration of immobility. Doses of 30 and 60 mg/kg had the opposite effects, i.e. they
Table 1 Effect of CF, CAF, AF, AEF, EF and EWF of the D. rugosa Poiret hydroalcoholic extract on open-field behavior in ratsa Treatment
Total locomotion
Rearing
Immobility time
% Central locomotion
Grooming time
Defecation
Control (12) CF (12) CAF (12) Control (14) AF (13) AEF (13) Control (51) EF (26) EWF (27)
107.8 9 12.5 118.1 9 9.4 115.1 99.1 116.9 910.0 118.5 99.4 150.9 911.4* 97.7 93.4 108.9 93.4* 112.7 96.0*
28.39 2.0 30.29 3.6 24.39 3.0 24.79 3.1 29.89 2.5 33.69 2.1* 21.9 9 1.1 25.39 1.6 23.8 9 2.4
17.79 6.3 9.6 92.3 19.295.8 21.69 7.3 9.49 2.0 4.59 1.0* 28.99 3.4 10.992.7* 14.7 92.7*
8.1 9 1.9 7.3 9 1.4 6.4 90.8 8.2 91.7 7.3 9 1.2 8.39 1.0 7.9 9 0.6 8.5 9 1.2 7.8 9 0.8
43.1 9 7.6 38.49 7.5 33.9 9 8.1 33.9 9 6.6 41.79 8.8 26.59 8.0 42.59 4.2 35.9 9 3.8 40.29 5.4
2.7 91.7 2.9 90.6 2.7 90.4 3.3 90.7 6.1 90.7* 4.2 90.8 2.6 90.3 2.8 90.4 2.6 90.4
Data are means 9S.E.M.; the number of animals is given in parenthesis. * PB0.05 — ANOVA/Tukey–Kramer test, different from the respective controls.
a
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
Fig. 3. Effect of D. rugosa Poiret hydroalcoholic extract (HE; 15 and 30 mg/kg, p.o.) and water (control) on entries into open (OAE) and closed arms (CAE) and total entries (TE) (A); time in the open (TOA) and closed arms (TCA) (B);% OAE and TOA (C) and elevated plus-maze behavior in rats. Data are means9 S.E.M.; the number of animals is given in parenthesis inside the bars. Different from the control (* PB 0.05, ** P B 0.01 — Kruskall–Wallis/Mann–Whitney’s U test).
tion and increased immobility time. The effects of these last two doses were not different, suggesting that 30 mg/kg have the maximum effect. There were no modifications in any of the other parame-
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ters assessed in the open-field at dose used, except for an increase in defecation observed with the dose of 60 mg/kg. The results with 15 mg/kg agree with the popular use of the plant as a stimulant (1–3). Concerning the fractions, AEF, EF and EWF shared the stimulant effect with 15 mg/kg of HE, observed on the same parameters of the openfield. An additional increase in rearing was observed only with AEF. The only effect of AF was an increase in defecation. The effect of HE 30 and 60 mg/kg and AF on defecation suggests an action on intestinal activity and supports the popular use of the plant as purgative (Le Cointe, 1934; Silva, 1935; Correˆa, 1984). The presence of saponins in the HE (Matheucci, 1996) may be related to the depressing effects on general activity. It has been described that some saponins decrease general activity by modifying dopamine receptors (Kim et al., 1995a,b, 1998). On the other hand, concerning the hyperactivity observed with HE 15 mg/kg, AEF, EF and EWF may assume that: (a) the mechanisms involved are not dopaminergic; (b) the saponins present are not those which modify the dopaminergic responses; or (c) the quantities of these substances in the extract and fractions are not sufficient to elicit the effects described. Indeed other neurotransmitters may be involved in present effects, we did not find any other relations between the chemical components of the plant and neurotransmitters, except for GABA and flavonoids (see below). Albeit two doses of HE (15 and 30 mg/kg) and all the fractions were tested in the EPM, only 15 mg/kg of HE was effective. An increase in number of total entries and a higher percentage of time spent in the open arm was recorded. This higher number of total entries agrees with the increase in locomotion observed in the open-field test. Longer time spent into the open arms (together with the higher percentage of central locomotion recorded in the open-field test) suggests an anxiolytic effect (Zangrossi et al., 1992; Silva et al., 1995, 1997; Nasello et al., 1998). This is the same dose that has been reported to protect from stressinduced gastric lesions (Matheucci, 1996). Flavonoids are present in the HE and several lines
L. Guaraldo et al. / Journal of Ethnopharmacology 72 (2000) 61–67
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Table 2 Effect of CF, CAF, AF, AEF, EF and EWF of the D. rugosa Poiret hydroalcoholic extract on elevated plus-maze behavior in ratsa Treatment
Total entries
Open arm entries
% Open arm entries Time in open arms
% Time in open arms
Control (12) CF (12) CAF (12) Control (14) AF (13) AEF (13) Control (45) EF (29) EWF (29)
7.5 91.4 8.891.2 9.7 91.4 7.0 91.1 6.4 90.9 9.6 91.1 5.5 90.5 6.0 90.8 6.8 90.8
3.99 1.0 4.09 0.8 4.49 0.9 2.79 0.7 2.39 0.7 4.19 0.9 2.39 0.3 2.99 0.5 3.49 0.5
37.4 98.1 43.3 9 3.5 45.8 96.5 30.1 95.7 26.4 97.1 38.1 96.4 33.3 94.1 43.6 95.0 43.9 94.9
17.8 95.5 16.6 93.6 23.1 97.9 10.5 93.0 6.5 92.9 15.5 93.8 13.3 93.1 18.7 94.5 18.6 94.1
a
53.4 916.5 49.9 910.8 69.3 9 23.8 31.4 99.1 21.2 9 8.5 50.0 9 11.5 39.8 99.4 56.0 9 13.5 55.7 9 12.3
Data are means 9S.E.M.; the number of animals is given in parenthesis.
of evidence show that natural and sinthetic flavonoids are potent anxiolytic agents without sedative, myorelaxant or amnestic effects. It is known the participation of GABA in these effects. (Haberlein et al., 1994; Marder et al., 1996; Medina et al., 1997). In conclusion, the present study showed that the HE of D. rugosa Poiret has a biphasic effect on general activity according to the dose used. The active compounds for the stimulant effects are in the three more polar fractions. Interestingly, the same dose that induces higher motor activity is anxiolytic. HE 30 and 60 mg/kg and AF may be also effective on intestinal activity. However, further studies including toxicologic evaluations and the use of more purified fractions are necessary to confirm and extend the present findings.
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