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Pharmacology of Casimiroa edulis IV. Hypotensive effects of compounds isolated from methanolic extracts in rats and guinea pigs
Journal of Ethnopharmacology 64 (1999) 35 – 44
Pharmacology of Casimiroa edulis IV. Hypotensive effects of compounds isolated from methanolic extracts in rats and guinea pigs Gil A. Magos a, Horacio Vidrio a,*, William F. Reynolds b, Rau´l G. Enrı´quez c a
Department of Pharmacology, School of Medicine, National Uni6ersity of Mexico, P.O. Box 70 -297, 04510 Mexico, DF, Mexico b Department of Chemistry, Uni6ersity of Toronto, Toronto, ON M5S 1A1, Canada c Institute of Chemistry, National Uni6ersity of Mexico, P.O. Box 70 -297, 04510 Mexico, DF, Mexico Received 5 November 1997; received in revised form 19 May 1998; accepted 19 May 1998
Abstract Bioassay-directed fractionation of the methanolic extract of seeds of Casimiroa edulis led to the isolation of seven constituents with cardiovascular activity, namely the new compound synephrine acetonide and the known compounds N-monomethylhistamine, N,N-dimethylhistamine, proline, N-methylproline, g-aminobutyric acid and casimiroedine. In anesthetized rats, both histamine derivatives produced transient hypotension mediated via H1-histaminergic receptors and in the case of N,N-dimethylhistamine, via nitric oxide release. Synephrine acetonide produced transient hypertension and tachycardia, mediated via a- and a- and b-adrenergic receptores, respectively. The chromatographic zone containing N-methyproline, proline and g-aminobutyric acid elicited marked and prolonged hypotension. Finally, casimiroedine did not modify the blood pressure of anesthetized rats, but lowered it persistently in anesthetized guinea pigs. It was concluded that hypotension produced by C. edulis is due to several active components. The immediate effect can be attributed to the histamine derivatives acting on H1-receptors. More prolonged hypotension would be produced by the mixture of amino acids through an unknown mechanism, as well as by casimiroedine, possibly by activation of H3-receptors. Hypotension is partially offset by synephrine acetonide through adrenergic mechanisms. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Casimiroa edulis; Hypotension; Histamine receptors; Adrenergic receptors; Nitric oxide; Amino acids
1. Introduction
* Corresponding author.
Casimiroa edulis de la Llave and Lexarza (Rutaceae), commonly known as ‘zapote blanco’ is a tree indigenous to Mexico, used by local herbal-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00101-9
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ists in the treatment of hypertension (Lozoya et al., 1987). The hypotensive activity of the plant was first recognized a hundred years ago (Anonymous, 1897) and later confirmed in animals (De Lille, 1934; Ramı´rez and Rivero, 1935; Lozoya et al., 1977; Magos and Vidrio, 1991) and humans (Flores Montalvo, 1936; Cruz y Corro, 1939). We recently found that the methanolic extract of ‘zapote blanco’ seeds exerts its hypotensive action apparently by peripheral vasodilation mediated through histamine H1-receptors and that it also produces a-adrenergic vasoconstriction (Magos and Vidrio, 1991; Vidrio and Magos, 1991; Magos et al., 1995). Hypotension produced by ‘zapote blanco’ was previously attributed solely to N,N-dimethylhistamine (Major and Du¨rsch, 1958; Lozoya et al., 1978), a histamine derivative present in the plant, which shares the hypotensive effects of the parent compound (Vartiainen, 1935; Bertaccini and Vitali, 1964). No detailed pharmacology of other possible vasoactive components of the seed has been described. In this work we report the hypotensive effects of several compounds present in the methanolic extract of ‘zapote blanco’ seeds, potentially jointly responsible for the action of the plant on blood pressure. Particular attention is given to the mechanism of action of the above mentioned histamine derivative, as well as of its N-monomethyl analog. Of the other compounds, some were previously reported for this plant and some are described for the first time. Detailed chemical work leading to the structural characterization of these compounds will be published elsewhere.
2. Materials and methods
2.1. Plant material Seeds of ripe fruits of C. edulis were collected in Texcoco, Mexico State. The botanical identity of the plant was verified and vouched at the herbarium of the Botanical Center of the Graduate College of Chapingo. The specimen deposited as Collection No. 1 of G.A. Magos, was authenticated by Jose´ Garcı´a Pe´rez, botanist in charge of the herbarium.
2.2. Sample preparation The dry ground kernels (1 kg) were extracted by maceration at room temperature with methanol. The solvent was eliminated by drying in vacuum and a portion of the extracted material (25 g) was subjected to column chromatography (CC) on silica gel 60 (500 g). Fractions obtained by discontinuous gradient elution with acetonitrile-methanol (60:40 v/v) and n-propanol-water (100:50 v/v), both in 1% ammonium hydroxide, were grouped on the basis of thin layer chromatography (TLC) and hypotensive activity. The active fractions were rechromatographed by CC and preparative TLC.
2.3. Compound identification The seven compounds found in the active fractions were identified by IR, NMR and MS spectra. IR spectra were obtained in Kbr or as a film with a Perkin-Elmer 283-B or a Nicolet FT-J.RSX spectrometer. 1H NMR spectra were obtained in methanol or D2O using a Varian XL 300 Mhz spectrometer. MS spectra were obtained with a Jeol JMS-SX102A mass spectrometer operating at 70 eV. Compounds in the fraction of higher polarity designated as the MP zone (see below) were identified by 1H and DEPT NMR spectra in conjuction with COSY, TOCSY, HMQC and HMBC NMR using a 500 Mhz instrument.
2.4. Preparation of anesthetized rats and guinea pigs Male Wistar rats weighing 250–300 g and male guinea pigs of mixed breed weighing 300–400 g, were anesthetized with sodium pentobarbital (30 mg/kg i.p.). After cannulation of the trachea for artificial respiration, polyethylene cannulas were inserted in a femoral artery and vein in rats or in a carotid artery and a jugular vein in guinea pigs, for continuous recording of blood pressure and drug administration, respectively. Mean arterial pressure (MAP) was recorded with a pressure transducer connected to the arterial cannula. The signal from the transducer was electronically dampened and inscribed on a Model 79 Grass
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polygraph. Heart rate (HR) was recorded with a tachograph driven by the undampened pulse signal of the transducer. In all experiments, groups of six animals were used and pretreatments were carried out i.v. 10 min before administration of the test compounds.
2.5. Drugs Micronized loratadine was supplied by Schering Plough, Mexico. The hydrochlorides of histamine, prazosin, DL-propranolol, L-phenylephrine and Nv-nitro-L-arginine methyl ester, as well as atropine sulfate, (9 )-synephrine and cimetidine free bases, g-aminobutyric acid, L-proline and N-methyl-L-proline were obtained from Sigma, St. Louis, MO. Loratadine and casimiroedine were used as resuspensions in distilled water; all other drugs were dissolved in isotonic saline solution. In all cases the volume injected was 1 ml/kg.
2.6. Statistical analysis Baseline MAP and HR values, as well as changes in these parameters in response to the test compounds at different doses or times were compared in control and pretreated groups by unpaired or paired Student’s t-test as indicated. Comparison of two treatment groups with the same control was carried out by analysis of variance followed by Dunnett’s test. A probability level of less than 0.05 was considered as indicating statistical significance of the differences observed.
3. Results The bioassay-guided fractionation of the methanolic extract of ‘zapote blanco’ seeds led to identification of a chromatographic zone from which several compounds with cardiovascular activity were isolated. These were identified as the histamine (H) derivatives N-momomethylhistamine (MMH) and N,N-dimethylhistamine (DMH), and the imidazolic glucoalkaloid casimiroedine (CAS), all producing hypotension, as well as the phenethylamine derivative synephrine acetonide (SA), which increased MAP
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and HR. Silica gel rechromatography of the methanolic extract produced in 0.09% yield a homogeneous fraction of higher polarity (MP zone) also eliciting hypotension. High field and bidimensional NMR revealed the presence of the amino acids N-methylproline (MP, 70%), proline (P, 18%), g-aminobutyric acid (GABA, 12%) and other unidentified components (B 1%). The structures of the seven compounds identified appear in Fig. 1.
3.1. Studies of histamine deri6ati6es Both MMH and DMH, as well as H, produced short-lasting, dose-related decreases in MAP (Fig. 2). The derivatives were respectively two and ten times less potent than H. Responses were significantly inhibited by the peripheral H1-antagonist loratadine but not by the H2-blocker cimetidine. In contrast to H, which did not modify HR at any dose, both derivatives elicited positive chronotropic effects which were not affected by H1- or H2-blockade. In another series, dose-response curves to H, MMH and DMH were obtained in rats receiving a continuous infusion of phenylephrine and subsequently pretreated with the nitric oxide synthesis inhibitor Nv-nitro-L-arginine methylester (LNAME). Phenylephrine significantly increased MAP from 1239 2 to 1559 2 mmHg and decreased HR from 42796 to 3959 8 beats/min. These values were not changed further after LNAME, MAP being 156 9 5 mmHg and HR 4009 7 beats/min. Hypotensive responses to H were similar to those of animals not receiving the vasoconstrictor, whereas around 10-fold higher doses of the derivatives were required to induce equivalent depressor effects. Additional pretreatment with L-NAME significantly antagonized responses to H and DMH, but not to MMH. In this model, H and its two analogs increased HR; this effect was inhibited by L-NAME only in the case of DMH (Fig. 3).
3.2. Studies of synephrine acetonide All experiments in this series were carried out after pretreatment with atropine, in order to block
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Fig. 1. Structure of active compounds isolated from Casimiroa edulis. Structures of the reference drugs histamine and synephrine, not present in the plant, are shown for comparison.
baroreflex-induced vagal bradycardia. The cyclized phenethylamine derivative SA produced transient, dose-related increases in MAP and HR identical to those elicited by the reference drug synephrine (S, Fig. 4). Pressor responses to both agents were significantly inhibited by the a-adrenergic antagonist prazosin, but were unaffected by the b-adrenergic blocker propranolol. In contrast, tachycardia after SA and S was partially inhibited by both antagonists.
3.3. Studies of the MP zone and casimiroedine The MP zone, tested in rats at a single dose of 10 mg/kg, elicited marked hypotension lasting more than 60 min, without affecting HR (Fig. 5A). The constituent amino acids, administered either separately or as a mixture, had no effects on MAP or HR (results not shown). The glucoalkaloid CAS lacked hypotensive activity in rats, but produced a prolonged fall in MAP, also with
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Fig. 2. Effects of histamine, N-monomethylhistamine and N,N-dimethylhistamine on mean arterial pressure (upper panels) and heart rate (lower panels) of anesthetized rats. Shown are dose-response curves obtained in control animals () and in animals pretreated with loratadine, 10 mg/kg i.v. () or cimetidine, 10 mg/kg i.v. (). Symbols are means 9S.E.M. of six rats. Asterisks denote significant differences from control, P B 0.05. Abscissae correspond to i.v. doses of the imidazoles; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
no appreciable change in HR, when tested in guinea pigs (Fig. 5B). It shoud be noted that baseline MAP in these animals was 679 1 mmHg, so that the 20 mmHg decrease observed after CAS corresponds to a 30% fall, i.e. similar to that produced in rats by the MP zone.
4. Discussion Of the seven compounds identified in the present study, CAS (Power and Callan, 1911) and DMH (Major and Du¨rsch, 1958) have been reported as constituents of C. edulis. MMH, MP, P and GABA are known compounds not previously described in the plant, while SA is an entirely new chemical entity. The identity of these active compounds was confirmed by comparison of their spectral parameters with those reported in the
literature (Panzica and Townsed, 1973; Lozoya et al., 1978; Toscano et al., 1997), as well as by TLC’s ran in parallel with standard samples. The new compound SA was also characterized by various physicochemical methods (Enrı´quez et al., unpublished). The present results show that in the rat, MMH and DMH elicit transient hypotensive responses lasting less than 3 min and entirely similar to those produced by H, thus confirming previous reports (Vartiainen, 1935; Bertaccini and Vitali, 1964). These compounds cannot therefore be solely responsible for the long-lasting decrease in MAP produced by Casimiroa extracts, a possibility which we have suggested previously (Magos and Vidrio, 1991). Results of experiments with H-antagonists indicate that hypotension is mediated by activation of peripheral, presumably vascular, H1-receptors; blockade by loratadine, an
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Fig. 3. Effects of histamine, N-monomethylhistamine and N,N-dimethylhistamine on mean blood pressure (upper panels) and heart rate (lower panels) of anesthetized rats receiving a continuous i.v. infusion of phenylephrine, 30 mcg/kg/min. The dose-response curves were obtained before () and after () pretreatment with L-NAME, 31 mg/kg i.v. Symbols are means 9 S.E.M. of six animals. Asterisks denote significant differences from responses before L-NAME, PB 0.05. Abscissae correspond to i.v. doses of the imidazoles; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
H1-antagonist which does not penetrate into the central nervous system (Simons and Simons, 1994), precludes involvement of central H1-receptors in this response. In contrast to H, which in the rat is devoid of effects on HR (Levi et al., 1982) and was so found in the present experiments, both MMH and DMH produced tachycardia. This response is apparently not mediated through H1- or H2-receptors, since it is not blocked by the corresponding antagonists. Stimulation of cardiac b-adrenegic receptors, such as that produced by high concentrations of H in rat atria (Cakici et al., 1992), could explain this finding. Since vascular relaxation by H in rat aortic rings is endothelium-dependent(van de Voorde and Leusen, 1983), the role of nitric oxide as the endothelium-derived relaxing factor in the hypotensive effects of H, MMH and DMH was
explored in rats pretreated with the nitric oxide synthesis inhibitor L-NAME. In preliminary experiments, even high doses of this agent did not affect depressor responses to H, a finding similar to that reported by others (Conrad and Whittemore, 1992; Nakahara et al., 1995). Blockade of hypotension induced by acetylcholine, another endothelium-dependent vasodilator, could be achieved by nitric oxide synthesis inhibition in rabbits previously subjected to vasoconstriction with phenylephrine (Rees et al., 1989). It was therefore reasoned that an increase in vascular tone would enhance nitric oxide release (Vargas et al., 1990) and amplify the consequences of nitric oxide synthesis inhibition. Experiments in phenylephrine-vasoconstricted rats show that hypotension induced by H and DMH, but surprisingly not by MMH, is nitric oxide-dependent. In agreement with this finding, preliminary results in
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Fig. 4. Effects of synephrine and synephrine acetonide on mean blood pressure (upper panels) and heart rate (lower panels) of anesthetized rats pretreated with atropine, 1 mg/kg i.v. Shown are dose-response curves obtained in animals pretreated with atropine alone (control), atropine+prazosin, 0.1 mg/kg i.v., and atropine+ propranolol, 1 mg/kg i.v. Symbols are means9 S.E.M. of six animals. Asterisks denote significant differences from control, P B0.05. Abscissae correspond to i.v. doses of the phenethylamine derivatives; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
rat aortic rigs indicate that removal of endothelium prevents relaxation by H and DMH, but not by MMH (Magos et al., unpublished). Taken together, these results suggest that vasodilatation by H and DMH is the result of stimulation of H1-receptors in vascular endothelium (Van de Voorde and Leusen, 1983), which leads to the release of nitric oxide and vascular smooth muscle relaxation. On the other hand, the action of MMH appears to be exerted on H1-receptors located in vascular smooth muscle. It should be noted that H elicits endothelium-independent vasodilatation in the rat femoral artery by activation of smooth muscle H receptors, although these have been identified as H2- (Krstic et al., 1991). On the other hand, H1-relaxation of dog mesenteric and gastroepiploic arteries has been attributed to release of prostaglandin I2 (Toda et al., 1982). In any event, further studies are necessary to elucidate the nature of MMH-induced vasodilatation.
Tachycardia produced by DMH, but not by H or MMH, was partially inhibited by L-NAME, suggesting involvement of nitric oxide in this response. In keeping with the above postulated b-adrenergic mechanism of DMH-induced tachycardia, L-NAME has been shown to decrease chronotropic responses to b-adrenergic stimulation (Reid et al., 1994), as well as to antagonize the facilitation by angiotensin of norepinephrine release from atrial sympathetic nerve endings (Gironacci et al., 1997). The cardiovascular effects of the new compound SA, i.e., transient hypertension and tachycardia, are identical to those of the reference drug S, which has been described as an adrenergic receptor agonsit with much less affinity for b1 than for a1 -receptors (Jordan et al., 1957; Brown et al., 1988). The a1- nature of the pressor effect of SA and S is clearly demonstrated by its block-
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Fig. 5. (A) Effects of the MP zone, 10 mg/kg i.v. () and saline, 1 ml/kg i.v. (), on mean arterial pressure (upper panel) and heart rate (lower panel) of anesthetized rats. (B) Effects of casimiroedine, 3.1 mg/kg i.v. () and saline, 1 ml/kg i.v. (), on mean arterial pressure (upper panel) and heart rate (lower panel) of anesthetized guinea pigs. In both cases, symbols are means 9S.E.M. of six animals. Abscissae correspond to time after injection; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
ade by the specific antagonist prazosin, an interaction found previously with crude Casimiroa extract in rat aortic rings (Magos et al., 1995). Their positive chronotropic action appears to be mediated by both a- and b-receptors, since it is partially antagonized by prazosin and propranolol. Tachycardia paradoxically produced by areceptor activation has been reported with phenylephrine, the m-OH isomer of S (Williamson et al., 1994). It is interesting that the hypotensive fraction of Casimiroa methanolic extract contains a compound eliciting an opposite effect on MAP, thus theoretically preventing an excessive depressor response to the extract. This constitutes another example of antagonistic actions of constituents of the same plant (Izaddosst and Robinson, 1991). In contrast to the transient hypotension produced by the H derivatives, the mixture of amino
acids designated as the MP zone elicits a long-lasting fall in MAP and could thus be responsible for the persistent hypotensive action of Casimiroa crude extract in the dog (Vidrio and Magos, 1991). Although peripherally administered GABA lowers blood pressure in the rat (Billingsley and Suria, 1982), its effects are short-lived and cannot account for the prolonged MAP response to the MP zone. The other major components, P and MP, are devoid of hypotensive activity either by themselves or as a mixture with GABA. The nature of the blood pressure effect of the MP zone remains unexplained, although it is possible that the amino acids found are in effect residues of a peptide responsible for this effect. In this connection, it should be mentioned that P is a constituent of the potent vasodilator peptides kallidin and bradykinin (Trifilieff et al., 1993).
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CAS, the main component of Casimiroa seeds (Kincl et al., 1956), is devoid of blood pressure effects in rats, but elicits long-lasting hypotension in guinea pigs and could therefore be another constituent of Casimiroa extract contributing to its depressor effect. Although in the present study the mechanism of CAS hypotension was not investigated, stimulation of histaminergic H3-receptors is an interesting possibility, considering the imidazolic nature of the compound. These receptors, located in sympathetic nerve terminals, inhibit the release of norepinephrine and thus lead to vasodilatation and lowering of blood pressure (McLeod et al., 1993). The lack of effect of CAS on the blood pressure of rats as compared to guinea pigs parallels the species differences found for the prototypic H3-agonist R-a-methylhistamine (McLeod et al., 1994). In conclusion, the present findings indicate that hypotension after C. edulis is due to several active components. Immediate, transitory effects can be attributed to MMH and DMH, acting through H1-histaminergic receptors located in vascular smooth muscle and vascular endothelium, respectively. More prolonged hypotension would be produced by a mixture of the amino acids MP, P and GABA through an unknown mechanism, as well as by CAS, possibly by activation of H3-histaminergic receptors. Coexisting with these hypotensive components is SA, which increases blood pressure and heart rate by a- and b-adrenergic mechanisms
Acknowledgements The authors wish to thank the National University of Mexico for financial support through projects IN214094 and FM-012314 of Direccio´n General de Asuntos del Personal Acade´mico and Coordinacio´n General de Estudios de Posgrado, respectively.
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Pharmacology of Casimiroa edulis IV. Hypotensive effects of compounds isolated from methanolic extracts in rats and guinea pigs Gil A. Magos a, Horacio Vidrio a,*, William F. Reynolds b, Rau´l G. Enrı´quez c a
Department of Pharmacology, School of Medicine, National Uni6ersity of Mexico, P.O. Box 70 -297, 04510 Mexico, DF, Mexico b Department of Chemistry, Uni6ersity of Toronto, Toronto, ON M5S 1A1, Canada c Institute of Chemistry, National Uni6ersity of Mexico, P.O. Box 70 -297, 04510 Mexico, DF, Mexico Received 5 November 1997; received in revised form 19 May 1998; accepted 19 May 1998
Abstract Bioassay-directed fractionation of the methanolic extract of seeds of Casimiroa edulis led to the isolation of seven constituents with cardiovascular activity, namely the new compound synephrine acetonide and the known compounds N-monomethylhistamine, N,N-dimethylhistamine, proline, N-methylproline, g-aminobutyric acid and casimiroedine. In anesthetized rats, both histamine derivatives produced transient hypotension mediated via H1-histaminergic receptors and in the case of N,N-dimethylhistamine, via nitric oxide release. Synephrine acetonide produced transient hypertension and tachycardia, mediated via a- and a- and b-adrenergic receptores, respectively. The chromatographic zone containing N-methyproline, proline and g-aminobutyric acid elicited marked and prolonged hypotension. Finally, casimiroedine did not modify the blood pressure of anesthetized rats, but lowered it persistently in anesthetized guinea pigs. It was concluded that hypotension produced by C. edulis is due to several active components. The immediate effect can be attributed to the histamine derivatives acting on H1-receptors. More prolonged hypotension would be produced by the mixture of amino acids through an unknown mechanism, as well as by casimiroedine, possibly by activation of H3-receptors. Hypotension is partially offset by synephrine acetonide through adrenergic mechanisms. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Casimiroa edulis; Hypotension; Histamine receptors; Adrenergic receptors; Nitric oxide; Amino acids
1. Introduction
* Corresponding author.
Casimiroa edulis de la Llave and Lexarza (Rutaceae), commonly known as ‘zapote blanco’ is a tree indigenous to Mexico, used by local herbal-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00101-9
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ists in the treatment of hypertension (Lozoya et al., 1987). The hypotensive activity of the plant was first recognized a hundred years ago (Anonymous, 1897) and later confirmed in animals (De Lille, 1934; Ramı´rez and Rivero, 1935; Lozoya et al., 1977; Magos and Vidrio, 1991) and humans (Flores Montalvo, 1936; Cruz y Corro, 1939). We recently found that the methanolic extract of ‘zapote blanco’ seeds exerts its hypotensive action apparently by peripheral vasodilation mediated through histamine H1-receptors and that it also produces a-adrenergic vasoconstriction (Magos and Vidrio, 1991; Vidrio and Magos, 1991; Magos et al., 1995). Hypotension produced by ‘zapote blanco’ was previously attributed solely to N,N-dimethylhistamine (Major and Du¨rsch, 1958; Lozoya et al., 1978), a histamine derivative present in the plant, which shares the hypotensive effects of the parent compound (Vartiainen, 1935; Bertaccini and Vitali, 1964). No detailed pharmacology of other possible vasoactive components of the seed has been described. In this work we report the hypotensive effects of several compounds present in the methanolic extract of ‘zapote blanco’ seeds, potentially jointly responsible for the action of the plant on blood pressure. Particular attention is given to the mechanism of action of the above mentioned histamine derivative, as well as of its N-monomethyl analog. Of the other compounds, some were previously reported for this plant and some are described for the first time. Detailed chemical work leading to the structural characterization of these compounds will be published elsewhere.
2. Materials and methods
2.1. Plant material Seeds of ripe fruits of C. edulis were collected in Texcoco, Mexico State. The botanical identity of the plant was verified and vouched at the herbarium of the Botanical Center of the Graduate College of Chapingo. The specimen deposited as Collection No. 1 of G.A. Magos, was authenticated by Jose´ Garcı´a Pe´rez, botanist in charge of the herbarium.
2.2. Sample preparation The dry ground kernels (1 kg) were extracted by maceration at room temperature with methanol. The solvent was eliminated by drying in vacuum and a portion of the extracted material (25 g) was subjected to column chromatography (CC) on silica gel 60 (500 g). Fractions obtained by discontinuous gradient elution with acetonitrile-methanol (60:40 v/v) and n-propanol-water (100:50 v/v), both in 1% ammonium hydroxide, were grouped on the basis of thin layer chromatography (TLC) and hypotensive activity. The active fractions were rechromatographed by CC and preparative TLC.
2.3. Compound identification The seven compounds found in the active fractions were identified by IR, NMR and MS spectra. IR spectra were obtained in Kbr or as a film with a Perkin-Elmer 283-B or a Nicolet FT-J.RSX spectrometer. 1H NMR spectra were obtained in methanol or D2O using a Varian XL 300 Mhz spectrometer. MS spectra were obtained with a Jeol JMS-SX102A mass spectrometer operating at 70 eV. Compounds in the fraction of higher polarity designated as the MP zone (see below) were identified by 1H and DEPT NMR spectra in conjuction with COSY, TOCSY, HMQC and HMBC NMR using a 500 Mhz instrument.
2.4. Preparation of anesthetized rats and guinea pigs Male Wistar rats weighing 250–300 g and male guinea pigs of mixed breed weighing 300–400 g, were anesthetized with sodium pentobarbital (30 mg/kg i.p.). After cannulation of the trachea for artificial respiration, polyethylene cannulas were inserted in a femoral artery and vein in rats or in a carotid artery and a jugular vein in guinea pigs, for continuous recording of blood pressure and drug administration, respectively. Mean arterial pressure (MAP) was recorded with a pressure transducer connected to the arterial cannula. The signal from the transducer was electronically dampened and inscribed on a Model 79 Grass
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polygraph. Heart rate (HR) was recorded with a tachograph driven by the undampened pulse signal of the transducer. In all experiments, groups of six animals were used and pretreatments were carried out i.v. 10 min before administration of the test compounds.
2.5. Drugs Micronized loratadine was supplied by Schering Plough, Mexico. The hydrochlorides of histamine, prazosin, DL-propranolol, L-phenylephrine and Nv-nitro-L-arginine methyl ester, as well as atropine sulfate, (9 )-synephrine and cimetidine free bases, g-aminobutyric acid, L-proline and N-methyl-L-proline were obtained from Sigma, St. Louis, MO. Loratadine and casimiroedine were used as resuspensions in distilled water; all other drugs were dissolved in isotonic saline solution. In all cases the volume injected was 1 ml/kg.
2.6. Statistical analysis Baseline MAP and HR values, as well as changes in these parameters in response to the test compounds at different doses or times were compared in control and pretreated groups by unpaired or paired Student’s t-test as indicated. Comparison of two treatment groups with the same control was carried out by analysis of variance followed by Dunnett’s test. A probability level of less than 0.05 was considered as indicating statistical significance of the differences observed.
3. Results The bioassay-guided fractionation of the methanolic extract of ‘zapote blanco’ seeds led to identification of a chromatographic zone from which several compounds with cardiovascular activity were isolated. These were identified as the histamine (H) derivatives N-momomethylhistamine (MMH) and N,N-dimethylhistamine (DMH), and the imidazolic glucoalkaloid casimiroedine (CAS), all producing hypotension, as well as the phenethylamine derivative synephrine acetonide (SA), which increased MAP
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and HR. Silica gel rechromatography of the methanolic extract produced in 0.09% yield a homogeneous fraction of higher polarity (MP zone) also eliciting hypotension. High field and bidimensional NMR revealed the presence of the amino acids N-methylproline (MP, 70%), proline (P, 18%), g-aminobutyric acid (GABA, 12%) and other unidentified components (B 1%). The structures of the seven compounds identified appear in Fig. 1.
3.1. Studies of histamine deri6ati6es Both MMH and DMH, as well as H, produced short-lasting, dose-related decreases in MAP (Fig. 2). The derivatives were respectively two and ten times less potent than H. Responses were significantly inhibited by the peripheral H1-antagonist loratadine but not by the H2-blocker cimetidine. In contrast to H, which did not modify HR at any dose, both derivatives elicited positive chronotropic effects which were not affected by H1- or H2-blockade. In another series, dose-response curves to H, MMH and DMH were obtained in rats receiving a continuous infusion of phenylephrine and subsequently pretreated with the nitric oxide synthesis inhibitor Nv-nitro-L-arginine methylester (LNAME). Phenylephrine significantly increased MAP from 1239 2 to 1559 2 mmHg and decreased HR from 42796 to 3959 8 beats/min. These values were not changed further after LNAME, MAP being 156 9 5 mmHg and HR 4009 7 beats/min. Hypotensive responses to H were similar to those of animals not receiving the vasoconstrictor, whereas around 10-fold higher doses of the derivatives were required to induce equivalent depressor effects. Additional pretreatment with L-NAME significantly antagonized responses to H and DMH, but not to MMH. In this model, H and its two analogs increased HR; this effect was inhibited by L-NAME only in the case of DMH (Fig. 3).
3.2. Studies of synephrine acetonide All experiments in this series were carried out after pretreatment with atropine, in order to block
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Fig. 1. Structure of active compounds isolated from Casimiroa edulis. Structures of the reference drugs histamine and synephrine, not present in the plant, are shown for comparison.
baroreflex-induced vagal bradycardia. The cyclized phenethylamine derivative SA produced transient, dose-related increases in MAP and HR identical to those elicited by the reference drug synephrine (S, Fig. 4). Pressor responses to both agents were significantly inhibited by the a-adrenergic antagonist prazosin, but were unaffected by the b-adrenergic blocker propranolol. In contrast, tachycardia after SA and S was partially inhibited by both antagonists.
3.3. Studies of the MP zone and casimiroedine The MP zone, tested in rats at a single dose of 10 mg/kg, elicited marked hypotension lasting more than 60 min, without affecting HR (Fig. 5A). The constituent amino acids, administered either separately or as a mixture, had no effects on MAP or HR (results not shown). The glucoalkaloid CAS lacked hypotensive activity in rats, but produced a prolonged fall in MAP, also with
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Fig. 2. Effects of histamine, N-monomethylhistamine and N,N-dimethylhistamine on mean arterial pressure (upper panels) and heart rate (lower panels) of anesthetized rats. Shown are dose-response curves obtained in control animals () and in animals pretreated with loratadine, 10 mg/kg i.v. () or cimetidine, 10 mg/kg i.v. (). Symbols are means 9S.E.M. of six rats. Asterisks denote significant differences from control, P B 0.05. Abscissae correspond to i.v. doses of the imidazoles; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
no appreciable change in HR, when tested in guinea pigs (Fig. 5B). It shoud be noted that baseline MAP in these animals was 679 1 mmHg, so that the 20 mmHg decrease observed after CAS corresponds to a 30% fall, i.e. similar to that produced in rats by the MP zone.
4. Discussion Of the seven compounds identified in the present study, CAS (Power and Callan, 1911) and DMH (Major and Du¨rsch, 1958) have been reported as constituents of C. edulis. MMH, MP, P and GABA are known compounds not previously described in the plant, while SA is an entirely new chemical entity. The identity of these active compounds was confirmed by comparison of their spectral parameters with those reported in the
literature (Panzica and Townsed, 1973; Lozoya et al., 1978; Toscano et al., 1997), as well as by TLC’s ran in parallel with standard samples. The new compound SA was also characterized by various physicochemical methods (Enrı´quez et al., unpublished). The present results show that in the rat, MMH and DMH elicit transient hypotensive responses lasting less than 3 min and entirely similar to those produced by H, thus confirming previous reports (Vartiainen, 1935; Bertaccini and Vitali, 1964). These compounds cannot therefore be solely responsible for the long-lasting decrease in MAP produced by Casimiroa extracts, a possibility which we have suggested previously (Magos and Vidrio, 1991). Results of experiments with H-antagonists indicate that hypotension is mediated by activation of peripheral, presumably vascular, H1-receptors; blockade by loratadine, an
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Fig. 3. Effects of histamine, N-monomethylhistamine and N,N-dimethylhistamine on mean blood pressure (upper panels) and heart rate (lower panels) of anesthetized rats receiving a continuous i.v. infusion of phenylephrine, 30 mcg/kg/min. The dose-response curves were obtained before () and after () pretreatment with L-NAME, 31 mg/kg i.v. Symbols are means 9 S.E.M. of six animals. Asterisks denote significant differences from responses before L-NAME, PB 0.05. Abscissae correspond to i.v. doses of the imidazoles; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
H1-antagonist which does not penetrate into the central nervous system (Simons and Simons, 1994), precludes involvement of central H1-receptors in this response. In contrast to H, which in the rat is devoid of effects on HR (Levi et al., 1982) and was so found in the present experiments, both MMH and DMH produced tachycardia. This response is apparently not mediated through H1- or H2-receptors, since it is not blocked by the corresponding antagonists. Stimulation of cardiac b-adrenegic receptors, such as that produced by high concentrations of H in rat atria (Cakici et al., 1992), could explain this finding. Since vascular relaxation by H in rat aortic rings is endothelium-dependent(van de Voorde and Leusen, 1983), the role of nitric oxide as the endothelium-derived relaxing factor in the hypotensive effects of H, MMH and DMH was
explored in rats pretreated with the nitric oxide synthesis inhibitor L-NAME. In preliminary experiments, even high doses of this agent did not affect depressor responses to H, a finding similar to that reported by others (Conrad and Whittemore, 1992; Nakahara et al., 1995). Blockade of hypotension induced by acetylcholine, another endothelium-dependent vasodilator, could be achieved by nitric oxide synthesis inhibition in rabbits previously subjected to vasoconstriction with phenylephrine (Rees et al., 1989). It was therefore reasoned that an increase in vascular tone would enhance nitric oxide release (Vargas et al., 1990) and amplify the consequences of nitric oxide synthesis inhibition. Experiments in phenylephrine-vasoconstricted rats show that hypotension induced by H and DMH, but surprisingly not by MMH, is nitric oxide-dependent. In agreement with this finding, preliminary results in
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Fig. 4. Effects of synephrine and synephrine acetonide on mean blood pressure (upper panels) and heart rate (lower panels) of anesthetized rats pretreated with atropine, 1 mg/kg i.v. Shown are dose-response curves obtained in animals pretreated with atropine alone (control), atropine+prazosin, 0.1 mg/kg i.v., and atropine+ propranolol, 1 mg/kg i.v. Symbols are means9 S.E.M. of six animals. Asterisks denote significant differences from control, P B0.05. Abscissae correspond to i.v. doses of the phenethylamine derivatives; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
rat aortic rigs indicate that removal of endothelium prevents relaxation by H and DMH, but not by MMH (Magos et al., unpublished). Taken together, these results suggest that vasodilatation by H and DMH is the result of stimulation of H1-receptors in vascular endothelium (Van de Voorde and Leusen, 1983), which leads to the release of nitric oxide and vascular smooth muscle relaxation. On the other hand, the action of MMH appears to be exerted on H1-receptors located in vascular smooth muscle. It should be noted that H elicits endothelium-independent vasodilatation in the rat femoral artery by activation of smooth muscle H receptors, although these have been identified as H2- (Krstic et al., 1991). On the other hand, H1-relaxation of dog mesenteric and gastroepiploic arteries has been attributed to release of prostaglandin I2 (Toda et al., 1982). In any event, further studies are necessary to elucidate the nature of MMH-induced vasodilatation.
Tachycardia produced by DMH, but not by H or MMH, was partially inhibited by L-NAME, suggesting involvement of nitric oxide in this response. In keeping with the above postulated b-adrenergic mechanism of DMH-induced tachycardia, L-NAME has been shown to decrease chronotropic responses to b-adrenergic stimulation (Reid et al., 1994), as well as to antagonize the facilitation by angiotensin of norepinephrine release from atrial sympathetic nerve endings (Gironacci et al., 1997). The cardiovascular effects of the new compound SA, i.e., transient hypertension and tachycardia, are identical to those of the reference drug S, which has been described as an adrenergic receptor agonsit with much less affinity for b1 than for a1 -receptors (Jordan et al., 1957; Brown et al., 1988). The a1- nature of the pressor effect of SA and S is clearly demonstrated by its block-
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Fig. 5. (A) Effects of the MP zone, 10 mg/kg i.v. () and saline, 1 ml/kg i.v. (), on mean arterial pressure (upper panel) and heart rate (lower panel) of anesthetized rats. (B) Effects of casimiroedine, 3.1 mg/kg i.v. () and saline, 1 ml/kg i.v. (), on mean arterial pressure (upper panel) and heart rate (lower panel) of anesthetized guinea pigs. In both cases, symbols are means 9S.E.M. of six animals. Abscissae correspond to time after injection; ordinates, to change from baseline blood pressure or heart rate in mmHg or beats/min, respectively.
ade by the specific antagonist prazosin, an interaction found previously with crude Casimiroa extract in rat aortic rings (Magos et al., 1995). Their positive chronotropic action appears to be mediated by both a- and b-receptors, since it is partially antagonized by prazosin and propranolol. Tachycardia paradoxically produced by areceptor activation has been reported with phenylephrine, the m-OH isomer of S (Williamson et al., 1994). It is interesting that the hypotensive fraction of Casimiroa methanolic extract contains a compound eliciting an opposite effect on MAP, thus theoretically preventing an excessive depressor response to the extract. This constitutes another example of antagonistic actions of constituents of the same plant (Izaddosst and Robinson, 1991). In contrast to the transient hypotension produced by the H derivatives, the mixture of amino
acids designated as the MP zone elicits a long-lasting fall in MAP and could thus be responsible for the persistent hypotensive action of Casimiroa crude extract in the dog (Vidrio and Magos, 1991). Although peripherally administered GABA lowers blood pressure in the rat (Billingsley and Suria, 1982), its effects are short-lived and cannot account for the prolonged MAP response to the MP zone. The other major components, P and MP, are devoid of hypotensive activity either by themselves or as a mixture with GABA. The nature of the blood pressure effect of the MP zone remains unexplained, although it is possible that the amino acids found are in effect residues of a peptide responsible for this effect. In this connection, it should be mentioned that P is a constituent of the potent vasodilator peptides kallidin and bradykinin (Trifilieff et al., 1993).
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CAS, the main component of Casimiroa seeds (Kincl et al., 1956), is devoid of blood pressure effects in rats, but elicits long-lasting hypotension in guinea pigs and could therefore be another constituent of Casimiroa extract contributing to its depressor effect. Although in the present study the mechanism of CAS hypotension was not investigated, stimulation of histaminergic H3-receptors is an interesting possibility, considering the imidazolic nature of the compound. These receptors, located in sympathetic nerve terminals, inhibit the release of norepinephrine and thus lead to vasodilatation and lowering of blood pressure (McLeod et al., 1993). The lack of effect of CAS on the blood pressure of rats as compared to guinea pigs parallels the species differences found for the prototypic H3-agonist R-a-methylhistamine (McLeod et al., 1994). In conclusion, the present findings indicate that hypotension after C. edulis is due to several active components. Immediate, transitory effects can be attributed to MMH and DMH, acting through H1-histaminergic receptors located in vascular smooth muscle and vascular endothelium, respectively. More prolonged hypotension would be produced by a mixture of the amino acids MP, P and GABA through an unknown mechanism, as well as by CAS, possibly by activation of H3-histaminergic receptors. Coexisting with these hypotensive components is SA, which increases blood pressure and heart rate by a- and b-adrenergic mechanisms
Acknowledgements The authors wish to thank the National University of Mexico for financial support through projects IN214094 and FM-012314 of Direccio´n General de Asuntos del Personal Acade´mico and Coordinacio´n General de Estudios de Posgrado, respectively.
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