Effects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve.
Alpinia zerumbet, known popularly as "colonia" in Northeastern Brazil, is a medicinal plant that has been used widely in folk medicine as teas and infusions for the treatment of intestinal and cardiovascular diseases, including arterial hypertension. Our previous studies have demonstrated that the essential oil of A. zerumbet (OEAZ) is very active on excitable tissues, such as smooth muscle, and in this study we verified its effects on the compound action potential (CAP) of rat sciatic nerve. EOAZ induced a dose-dependent blockade of the CAP. Control peak-to-peak amplitude and conduction velocity of CAPs were 7.6[+ or -]0.43 mV and 80.6[+ or -]3.19 m/s, respectively. At 60 [micro]g/ml, EOAZ induced no demonstrable effect. Conduction velocity was significantly reduced at 180 min of preparation exposure to 100 [micro]g/ml of EOAZ. At 300, 600 and 2000 [micro]g/ml doses of EOAZ, the peak-to-peak amplitudes of CAPs following 180 min exposure of the nerve to the drug were reduced significantly, to 75.3[+ or -]7.36%, 50.45[+ or -]2.17% and 0% respectively, of control value. Conduction velocity was reduced significantly by 300, 600 and 2000 [micro]g/ml of EOAZ, at 180 min, to 83.61[+ or -]3.28%, 64.06[+ or -]8.21% and 22.7[+ or -]5.79%, respectively, of control value. All these effects developed slowly and were reversible upon a 180-min wash.
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Keywords: Essential oil; Alpinia zerumbet; Alpinia speciosa; Sciatic nerve; Compound action potential
Plants of the genus Alpinia have been used widely all over the world in folk medicine (Itokawa et al., 1981a b, 1987), as protection from gastric lesions (Hsu, 1988), diuretic (Laranja et al., 1991, 1992), and as an antifungal (Klayman, 1985; Janssen and Scheffer, 1985). Alpinia zerumbet (Pers.) Burtt. Et Smith, also called Alpinia speciosa K. Schum., and known popularly as "colonia" in Northeastern Brazil, is a plant with wide use in folk medicine, as teas and infusions, for the treatment of intestinal and cardiovascular diseases, including arterial hypertension (Mendonca et al., 1991; Prudent et al., 1993; Bezerra et al., 2000).
Previous studies on A. zerumbet have demonstrated that its extracts have many biological activities, including inhibition of porphyrin photooxidative reaction (Liao et al., 2000) and antifungal (Lima et al., 1993), diuretic and antihypertensive (Laranja et al., 1991, 1992; Mendonca et al., 1991) properties. Among the extracts, the essential oil of A. zerumbet (EOAZ) was very effective and potent in regulating arterial pressure (Bezerra, 1994), inducing hypothension. EOAZ was also very active on smooth muscle (Bezerra et al., 2000) as an antispasmodic agent.
Because EOAZ acts on important physiological parameters, such as arterial pressure and muscle contractility, and because many essential oils and their chemical constituents are known to have potent local anaesthetic activity (Brodin and Roed, 1984a b; Dallmeier and Carlini, 1981; Ghelardini et al., 1999) the question naturally arises whether EOAZ acts on an important type of excitable cell, the neuron. To answer that question, the present investigation was undertaken on the rat sciatic nerve, which has motor and sensorial fibers. This study demonstrates that EOAZ depresses the amplitude and conduction velocity of the compound action potential (CAP) of the sciatic nerve.
Materials and methods
Extraction and administration of EOAZ
The essential oil was extracted from leaves of A. zerumbet, collected in areas near Cascavel city, Ceara State, Brazil, during August 2000. A voucher specimen of A. zerumbet has been deposited at Herbarium Prisco Bezerra (# 10858), as identified by Drs. Edson Paula Nunes and Peres Martins. The isolation of the essential oil was carried out at the Department of Organic and Inorganic Chemistry of the Federal University of Ceara, according to the method described by Craveiro et al. (1976). Briefly, freshly chopped plant leaves were placed in a glass flask, connected at one end to a glass vessel with water and at the other end to a water-cooled condenser. The water was heated to boiling, and the steam percolated through the chopped plant leaves and collected in the condenser. After condensation, the watery phase with its solutes, here called "hydrolate", separated from an oily phase, the essential oil, which, when rediluted in water on occasion is called the "pseudo-hydrolate". The composition of EOAZ was determined by gas chromatography and mass spectrometry. It contained (%): 1.8-cineole, 20.57; terpinen-4-ol, 19.39; [gamma]-terpinene, 15.08; sabinene, 9.68; p-cimene, 8.54; [alpha]-tujene, 6.35; [alpha]-terpinene, 3.88; [beta]-pinene, 3.02; limonene, 2.64; [alpha]-pinene, 2.38; terpinolene, 1.93; [beta]-mircene, 1.20; trans-cariophilene, 1.11; [alpha]-terpineol, 0.86; not identified, 3.35.
Nerve dissection, electrical recordings and protocols
Sciatic nerves were dissected from male Wistar rats (Ratus norvegicus; 300-350 g each). Rats were obtained from the HEMOCE (Hemotherapy Center of Ceara) vivarium and maintained at the vivarium of the Laboratory of Electrophysiology of Universidade Estadual do Ceara during at least the four days prior to the experiment with water and food ad libitum and control of light/dark hours (approximately 12/12). They were scarified by cervical dislocation, because anaesthesia could interfere with results. The nerve was mounted in a moist chamber (Theophilidis and Pavlidou, 1993) and one of its ends was stimulated at a frequency of 0.2 Hz, with electric pulses of 50-100-[micro]s duration at 10-20 V, delivered by an stimulus isolation unit (Model SIU4678, Grass Instruments Co., Quincy, MA, USA) connected to a stimulator (Model S48, Grass Instruments Co.). Evoked CAPs were recorded using platinum electrodes, placed 4 5 cm from the stimulating electrodes. For continuous monitoring, these were connected through a high-input impedance amplifier (Model P15, Grass Instruments CO.) to an oscilloscope (Model 547, Tektronix, Inc., Portland, OR, USA). For data capture and analysis, Digidata 1200 computer acquisition hard-ware (Axon Instruments, Inc., Foster City, CA, USA) and AxoScope software (Axon Instruments, Inc.) were used. A 15-20-mm segment of the nerve suspended between the stimulating and recording electrodes was immersed in Locke's solution, which was employed to maintain chamber humidity. Pharmacological agents were dissolved in Locke's solution and administered via the bath. Nerves were usually employed on the day of dissection; however, in some experiments they were stored overnight in cold (5[degrees]C) Locke's solution for use the following day. This storage period did not affect electrophysiological control parameters of the nerves.
Before exposure to drugs, the nerves were allowed to stabilize until stable peak-to-peak CAP amplitude recording was achieved for at least 30 min. Afterwards, the bathed nerve segment was exposed to pharmacological agents for 180 min. This interval was usually sufficient to allow steady-state action potential amplitude to be reached during EOAZ administration. This period was followed by wash and a 180-min recovery period. Experiments were performed at room temperature (24-26[degrees]C).
Solutions and drugs
Modified Locke's solution (composition in mM: 140 NaCl, 5.6 KCl, 2.2 Ca[Cl.sub.2], 1.2 Mg[Cl.sub.2], 10 Glucose, and 10 TRIS-(Hydroxymethyl)Aminomethane) (pH 7.4) was thoroughly aerated before use in the chamber. EOAZ stock solution [(vehicle, dimethylsulfoxide (DMSO)], prepared daily, was added to the chamber Locke's solution so as to provide the desired EOAZ concentration with a vehicle final concentration always lower than 0.25%, v/v. At such a concentration, DMSO did not alter CAP parameters. All salts and drugs, except for EOAZ, were purchased from Sigma Chemical (St. Louis, MO, USA) or Reagen (Rio de Janeiro, RJ, Brazil) and were of analytical grade.
Results are presented as the mean[+ or -]s.e. of mean, with (n) indicating the number of experiments. Values were analyzed using the Student's t test, or ANOVA followed by a contrast test, or a non-parametric test, as appropriate. Results were considered significant at p<0.05.
CAP peak-to-peak amplitude and conduction velocity in the control group (vehicle only), at the end of two hours' stabilization period, were 7.55[+ or -]0.43 mV and 80.6[+ or -]3.19 m/s (n = 10), respectively. In six experiments the preparation was, after the stabilization period, maintained for six ensuing hours in the presence of vehicle only and, at the end of this period, peak-to-peak amplitude and conduction velocity were 101.80[+ or -]4.91% and 98.7[+ or -]4.45%, respectively, of initial control values, showing good stability during that period.
[FIGURE 1 OMITTED]
During 180 min in the presence of 300, 600 or 2000 [micro]g/ml CAP, peak-to-peak amplitudes decreased monotonically (Figs. 1 and 2) and, at minute 180 they were significantly reduced to 75.26[+ or -]7.36%, 50.45[+ or -]2.17%, and 0%, respectively, of the control value. During 180 min in the presence of 100 [micro]g/ml, CAP peak-to-peak amplitude initially increased (without reaching significance) during the first 15 min and afterwards progressively decreased to approach control value at the minute 180. At a concentration of 60 [micro]g/ml, EOAZ did not alter the CAP (Fig. 2).
During 180 min in the presence of 300, 600 and 2000 [micro]g/ml, CAP conduction velocity decreased monotonically (Fig. 2) and, at minute 180, they were significantly (p<0.05, ANOVA and Student-Newman-Keuls method) reduced to 83.61[+ or -]3.28%, 64.1[+ or -]8.21% (n = 6), respectively, of the control value. In the presence of 2000 [micro]g/ml EOAZ, CAP conduction velocities decreased rapidly to 22.7[+ or -]5.79% (n = 4) of the control at 60 min, the last moment to allow resolution on measurement. At 100 [micro]g/ml EOAZ (Fig. 2), by contrast to peak-to-peak amplitude, conduction velocities mean values decreased monotonically to be significantly different from the control from minute 120 forward. After wash, CAP parameters increased during the 180-min recovery period (Fig. 2) to reach values not significantly different from the control (ANOVA and Student Newman-Keuls method; n = 6).
[FIGURE 2 OMITTED]
EOAZ-induced blockade of the CAP of rat sciatic nerve is the major discovery of the present study. To the best of our knowledge, there are no published studies dealing with the effects of EOAZ on parameters related to nerve excitability. This is thus a novel finding.
Despite widespread use of A. zerumbet in folk medicine (Itokawa et al., 1981a b, 1987) and the demonstration that EOAZ is pharmacologically active (Laranja et al., 1991, 1992; Mendonca et al., 1991; Bezerra et al., 2000), this essential oil has received very little attention from the scientific community. It has been shown that EOAZ is active in the cardiovascular system and in intestinal smooth muscle (Bezerra, 1994; Bezerra et al., 2000). Its intestinal effect was independent of action on the enteric nervous system (Bezerra et al., 2000). This study enlarges the list of pharmacological investigations with EOAZ available in the literature, because it demonstrates the effect of relatively small concentrations of EOAZ on an important excitable tissue, a peripheral nerve. It suggests the hypothesis that EOAZ is a local anaesthetic. It will, however, be necessary to demonstrate that EOAZ acts directly on the cytoplasmic membrane sodium channel, and this task awaits further investigation.
1.8-cineole is an important constituent of EOAZ, and this substance is said to be a mild local anaesthetic and antimicrobial agent (Craveiro et al., 1981) used in mouth and throat infections. The EOAZ-induced depressive effect on CAP is in agreement with that notion of 1,8-cineole anaesthetic effect. Another important constituent of EOAZ, terpinen-4-ol, has not been investigated for local anaesthetic or nerve excitability blocking effect. A substance with similar molecular structure, [alpha]-terpineol, has however been reported to be an important depressant of nerve excitability (Moreira et al., 2001). The effect on CAP of another monoterpenoid, eugenol, with a molecular structure similar to those of 1,8-cineole and terpinen-4-ol, has been documented to block the CAP of the sciatic nerve (Brodin and Roed, 1984a b). Regarding the EOAZ-induced depression of peripheral nerve excitability demonstrated here, it is important to mention that this study used a preparation and a protocol designed to study inhibition of excitability. It does not exclude the possibility of other types of EOAZ-induced neural actions. For instance, 1,8-cineole is a known stimulant of olfactory receptors (Reisert and Matthews, 2001; Bonviso and Chaput, 2000; Firestein et al., 1993), an effect likely to be shared by EOAZ.
Two aspects of the time course of EOAZ action deserve consideration. First, CAP blockade developed slowly: 3 h of preparation exposure to this essential oil, although a long enough time do demonstrate significant effect for 300 and 600 [micro]g/ml, were not enough to establish steady-state phase of effect (we opted for only 3 h of exposure to EOAZ to leave enough time to demonstrate preparation recovery after wash, which was also slow). This suggests that the maximal effects of these concentrations of EOAZ on CAP are larger than those reported here. It also raises the possibility that EOAZ might act slowly in sites other than the membrane, perhaps intracellularly, leading indirectly to perturbation of membrane function. Eugenol, whose molecule bears similarity to 1,8-cineole and terpinen-4-ol, is known to affect intracellular levels of ATP and glutathione. In a previous study we demonstrated that EOAZ, at the same concentration range used for the nerve, has a relaxant and antispasmodic activity on intestinal smooth muscle (Bezerra et al., 2000) that was independent of neural participation. Because in that study, the time of preparation exposure to EOAZ lasted less than one and half hour, the effect on neural components might not have had enough time to develop. The second factor to consider is that 100 [micro]g/ml EOAZ initially induced an increase in CAP peak-to-peak amplitude. We did not investigate the cause of that. Should it be proved significant, however, by increasing the number of observations it seems unlikely that it represents an increase in excitability, since velocity of conduction was not increased.
Control values for both parameters used here to quantify CAP, conduction velocity and peak-to-peak amplitude, are well within the range reported by others in peripheral nerve recordings in vitro and in vivo. Our control conduction velocities (81 m/s) in the upper range reported by others (100-16 m/s (Moreira et al., 2001; Miyoshi and Goto, 1973; Petran, 1967; Zachar, 1967)), reflect the good condition of our preparations. The preparation showed great and long-lasting (approximately 8 h) stability, which also confirms the good physiological quality of our preparations.
In conclusion, we have here demonstrated that EOAZ, in concentrations less than the 1 mg/ml range, is active on the nerve, blocking CAP generation and conduction. This finding is relevant on its own, due to the potential pharmacological importance of essential oils. It is also important to show that A. zerumbet has active components, such is its essential oil. Because this plant has received widespread use in folk medicine, our report implies the necessity of more studies on the extracts of this plant.
Received 17 June 2003; accepted 29 July 2003
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J.H. Leal-Cardoso*, M.R. Moreira, G.M. Pinto da Cruz, S.M. de Morais, M.S. Lahlou, A.N. Coelho-de-Souza
Centro de Ciencias da Saude, Universidade Estadual do Ceara, Av. Paranjana 1700 Campus Itaperi 60,740-000, CEP, Fortaleza, CE. Brazil
*Corresponding author: Tel.: +55-85-299-2714.
E-mail address: email@example.com (J.H. Leal-Cardoso).
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|Author:||Leal-Cardoso, J.H.; Moreira, M.R.; Pinto da Cruz, G.M.; de Morais, S.M.; Lahlou, M.S.; Coelho-de-Sou|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 1, 2004|
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