Extracts from plants used in Mexican traditional medicine activate [Ca.sup.2+]-dependent chloride channels in Xenopus laevis oocytes.
The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 [micro]g/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium morale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 [+ or -] 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 [micro]M), suggesting that the currents were dependent on intracellular [Ca.sup.2+] increase. Thus the plant extracts activate the typical oscillatory [Ca.sup.2+]-dependent CI- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of [Ca.sup.2+] from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.
Key words: Baccharis heterophylla, Chenopodium morale, Desmodium grahami, Gentiana spathacea, Solanum rostratum, Xenopus oocyte, [Ca.sup.2+]-dependent [Cl.sup.-] current
Our previous studies have shown that the chloroform:methanol (1:1) extracts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium morale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produce a concentration-dependent inhibition of spontaneous ileum contractions (Rojas et al., 1999). The smooth-muscle-relaxing activity displayed by the extracts supports the ethnomedical use of these plants in the state of Queretaro (Mexico) for alleviating gastrointestinal disorders. Recently, we started employing the Xenopus laevis oocyte as a cellular model to evaluate the effect of crude extracts derived from the above mentioned plants. It is expected that this model will allow us to extend the scope of bioassays that could be used to detect the pharmacological activity of plant extracts, to guide fractionation procedures and to help to assess the mechanism of action of isolates.
Native Xenopus oocytes and follicles, i.e. the oocytes with their surrounding follicular cells, are a very useful cellular model for studying neurotransmitter and hormone receptors, ionic channels and the intracellular mechanisms that operate them (for reviews see, Miledi et al., 1989; Arellano et al., 1996). More over, several exogenous receptors and channels can be expressed in the membrane of the oocyte by microinjection of messenger RNA from different sources (Miledi et al., 1989). This makes Xenopus oocytes and follicles excellent tools for the development and testing of new drugs.
A large number of medicinal plants have not yet been studied in detail for their pharmacological activities, and the Xenopus oocyte model offers the possibility of increasing our knowledge of biologically active constituents of plants in the search for novel drug molecules. Therefore, in this study we selected plants used in Mexican traditional medicine, with proven smooth-muscle-relaxing properties, as good candidates for further analysis.
Conventional studies on the chemical composition of some of these species have been described previously; however, neither the plant extracts nor the constituents have been studied electrophysiologically. Phytochemical investigations of B. heterophylla have allowed the isolation of different types of secondary metabolites, including several flavonoids (Wollenweber et al., 1986) and some terpenoids (Arriaga-Giner et al., 1986). Gentiopicroside has been isolated from G. spathacea (Rojas et al., 2000) and C. murale was found to contain some flavonoids (El Sayed et al., 1999; Gohar and Elmazar, 1997).
* Materials and Methods
Plant material and preparation of extracts The plant materials used in the present study were collected at the localities of Amealco and Toliman, State of Queretaro (Mexico), in June 1998. Authenticated Voucher specimens (B. heterophylla voucher no. Serrano 28; C. murale voucher no Serrano 543, D. grahami voucher no. Serrano 14; G. spathacea voucher no. Serrano 72 and S. rostratum voucher no. Serrano 558) were deposited in the Ethnobotanical Collection of the Herbarium of Queretaro "Dr. Jerzy Rzedowski" (QMEX), located at the School of Natural Sciences, University of Queretaro.
Coarsely-powdered, shade-dried aerial parts of the plants (100 g) were extracted exhaustively by maceration with 500 ml of a solvent mixture of C[H.sub.3]OH-CH[Cl.sub.3] (1:1) at room temperature. The resulting extracts were concentrated in vacuo and stored at room temperature for later use. For pharmacological assays, all extracts were dissolved in dimethylsulfoxide (DMSO).
Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI). Small pieces of ovarian lobes were dissected out from frogs, anesthetized with 1 g/l with tricaine and rendered hypothermic. The ovarian lobes were placed in sterile normal Barth's solution (SNBS) (mM: 88 NaCl, 1 KCl, 0.4 Ca[Cl.sub.2], 0.33 Ca[(N[O.sub.3]).sub.2], 0.82 MgS[O.sub.4], 2.4 NaHC[O.sub.3], 5 HEPES, pH 7.4 with 70 [micro]g/ml gentamicin). Follicles containing oocytes at stages V and VI (Dumont, 1972) were plucked from the ovaries and the follicular cells surrounding the oocytes were removed by collagenase treatment (0.5 mg/ml, 30 min at room temperature) in normal Ringer solution (NR, containing in mM: 115 NaCl, 2 KCl, 1.8 Ca[Cl.sub.2] and 5 HEPES, pH 7.0). Any remaining follicular layers were removed using sharp forceps. Defolliculated oocytes were incubated (18-20[degrees]C) in SNBS until their use in electrophysiological studies, which were performed during the first week.
Oocytes were placed in a small chamber and perfused continuously with NR solution at room temperature. The oocyte membrane current was recorded using the two-electrode voltage-clamp technique (Miledi, 1982). Unless otherwise stated, the membrane potential was held at -60 mV for current recording. Extracts, previously dissolved in DMSO, were diluted in NR (total volume of 20 ml and final concentration of DMSO [less than or equal to] 0.1%) and applied to the oocytes by continuous superfusion (10 ml/min). Experiments with 1,2-bis (2-aminophenoxy) ethane-N,N,N', N'-tetra acetic acid tetra(acetomethoxymethyl)ester (BAPTA-AM) were performed in oocytes incubated overnight with the drug.
BAPTA-AM was purchased from Molecular Probe (Eugene, Or. USA.).
Collagenase type I, tricaine, DMSO, Hepes and salts were obtained from Sigma Chemical Co. (St Louis, Mo. USA).
* Results and Discussion
The oocytes used in this study had a mean resting potential of -33 [+ or -] 1.8 mV (82 oocytes, 7 frogs) (all data given as means [+ or -] S. E.) and a mean input resistance of 0.904 [+ or -] 0.02 M[OMEGA]. The oocytes, held at -60 mV, responded to applications of the CH[Cl.sub.3]-MeOH (1:1) extracts of B. heterophylla, C. murale, D. grahami and S. rostratum with oscillatory inward currents (Fig. 1). The ion currents generated were in the range of 40 to 1600 nA. Subsequent extract applications to a single oocyte produced desensitization of the response. Therefore, the concentration-response curves were obtained by perfusing a given extract at one concentration for 2 min and washing for 20 min, before perfusing with a higher concentration. Fig. 2 shows the concentration-response curves for the four extracts. It is necessary to note that a clear maximal response was not obtained, since greater concentrations of DMSO (>0.1%) produce secondary effects on the oocyte membrane and it was not possible to test higher extract concentrations. Thus, the apparent values of the half-maximal effective concentration (E[C.sub.50] value) and potencies of the extracts are presented in Table 1. It seems that the extracts of D. grahami (E[C.sub.50] value = 23.97 [micro]g/ml) and S. rostratum (E[C.sub.50] value = 31.76 [micro]g/ml) were more potent than those from B. heterophylla (E[C.sub.50] value = 60.36 [micro]g/ml) and C. murale (E[C.sub.50] value = 79.40 [micro]g/ml), while the extract of G. spathacea was completely inactive. It is worth noting that preliminary evaluations of gentiopricroside, the major active principle of G. spathacea (Rojas et al., 2000), have shown that this compound did not produce any response in oocytes at concentrations up to 500 mg/ml (unpublished observations, A. Rojas and R. O. Arellano).
[FIGURES 1-2 OMITTED]
Current-voltage relationships for the oscillatory inward currents were obtained in oocytes voltage-clamped at different potentials (from -60 to 0 mV) and then superfused with the extracts (20 [micro]g/ml). The peak amplitude of the currents elicited by the extracts at every clamped potential was plotted (Fig. 3). The oscillatory inward currents induced by the plant extracts showed reversal potentials with an average of -17 [+ or -] 2 mV (30 oocytes, 5 frogs), which corresponded to the chloride equilibrium potential in Xenopus oocytes (Kusano et al., 1982), indicating that the currents were carried mainly by chloride ions.
[FIGURE 3 OMITTED]
It has been shown that several membrane receptors are present in the oocyte; for instance, the receptors to acetylcholine, angiotensin II, receptors to a serum factor, and to divalent cations (Miledi et al., 1989). Activation of these receptors by their respective agonists elicits well-characterized oscillatory [Ca.sup.2+]-dependent chloride currents in the oocyte membrane.
To study whether the oscillatory chloride current produced by the plant extracts was dependent on the increase of intracellular [Ca.sup.2+] concentration, we monitored the elicited responses in oocytes loaded overnight with BAPTA-AM (10 [micro]M), a calcium chelator. The oscillatory currents induced by every extract (20 [micro]g/ml) were abolished completely in the BAPTA-loaded oocytes (20 oocytes, 4 frogs), suggesting that their generation was strictly dependent on intracellular [Ca.sup.2+] (Fig. 4). In clear contrast, the responses were not affected when extracellular [Ca.sup.2+] was substituted by [Mg.sup.2+] (5 mM) in the NR solution (25 oocytes, 5 frogs) These findings indicated that the responses were not dependent on extracellular calcium influx.
[FIGURE 4 OMITTED]
The oscillatory chloride currents induced by other agonists in the oocyte membrane are due to mobilization of intracellular [Ca.sup.2+] by inositol polyphosphates and opening of [Ca.sup.2+]-gated chloride channels present in the oocyte membrane (Miledi, 1982; Miledi and Parker, 1984; Oron et al., 1985; Parker and Miledi, 1986). The results derived from this study strongly suggest that the [Ca.sup.2+]-dependent chloride currents evoked by the plant extracts are originated by activation of similar ionic channels. Further experiments will, however, be necessary to obtain information concerning the level at which the extracts activate the [Ca.sup.2+] release, and to determine if the I[P.sub.3]-diacylglycerol pathway is activated in Xenopus oocytes exposed to the extracts. The complete transduction system comprises several steps including: receptor stimulation, activation of phospholipase C via a G protein, and the subsequent production of I[P.sub.3]-diacylglycerol and raising of intracellular [Ca.sup.2+] concentration that opens the [Ca.sup.2+]-dependent chloride channels. The extracts might be acting at any of these levels.
At this stage of the study, elucidation of the exact mechanism of action of the extracts is hampered by the fact that they are complex mixtures which may contain hundreds of different metabolites. Moreover, very little is known about the chemical nature and/or the pharmacological properties of the constituents of these active plants. For instance, it has been reported that flavonoids are the main components of B. heterophylla (Wollenweber et al., 1986) and C. murale (El Sayed et al., 1999; Gohar and Elmazar, 1997). As far as we know, however, there is no evidence that suggests that these compounds might be interacting with any of the receptors, enzymes and/or channels present in the oocyte membrane. In the cases of D. grahami and S. rostratum, there is no information about their chemical composition. Most of the oocytes tested did not present responses to stimulation of receptors by the different agonists mentioned previously (e.g., acetylcholine, angiotensin II) but responded strongly to the extracts. Thus, it seems that the active substances in the extracts did not stimulate a known native membrane receptor. The results obtained from the present research allowed us to detect, in a simple cellular model, the activity of extracts from promising species which are now good candidates to pursue bioactivity-guided fractionation and purification of the active principles.
The results derived from this electrophysiological study provided relevant and new information concerning the pharmacological properties of the plants. We found that the extracts of B. heterophylla, C. murale, D. grahami and S. rostratum generated [Ca.sup.2+]-dependent chloride membrane currents in the Xenopus oocyte, apparently via [Ca.sup.2+] release from intracellular reservoirs.
Table 1. E[C.sub.50] value and potency ratios of the plant extracts. Extract E[C.sub.50] value Potency ([+ or -] S.E.) ([micro]g/ml) D. grahami 23.97 [+ or -] 8.52 1.00 S. rostratum 31.76 [+ or -] 8.04 0.75 B. heterophylla 60.36 [+ or -] 22.24 0.39 C. murale 79.40 [+ or -] 34.65 0.30 Potency was obtained using the formula: E[C.sub.50] value D. grahami/E[C.sub.50] value plant extract
This research was financed partially by Consejo Nacional de Ciencia y Tecnologia (CONACYT, Proyecto No. 25762-N and No I32862-N), Secretaria de Educacion Publica (FOMES 98-23-04) and Consejo de Ciencia y Tecnologia del Estado de Queretaro (CONCYTEQ). R. O. Arellano acknowledges grants from UNAM-DGAPA (IN-200198) and CONACYT (3713-PN). We are grateful to Dr. Edith Garay for technical assistance and to M. Sc. Valentina Serrano for collection and identification of plant material.
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A. Rojas, Facultad de Quimica, Universidad Autonoma de Queretaro, Centro Universitario, Queretaro, C.P. 76010, Mexico. Tel/Fax: ++524-2163730; e-mail: firstname.lastname@example.org
A. Rojas (1), S. Mendoza (1), J. Moreno (1), and R. O. Arellano (2)
(1) Facultad de Quimica, Universidad Autonoma de Queretaro, Centro Universitario, Queretaro, Mexico
(2) Centro de Neurobiologia, Universidad Nacional Autonoma de Mexico, Queretaro, Mexico