ACE activity during the hypotension produced by standardized aqueous extract of Cecropia glaziovii Sneth: a comparative study to captopril effects in rats.

Abstract

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8 [+ or -] 2.6 nmol/min/ml), intermediate ACE (39.4 [+ or -] 1.4 nmol/min/ml) and low ACE (23.5 [+ or -] 0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7 [+ or -] 0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Angiotensin converting enzyme; Hypotension; Cecropia glaziovii; Phytomedicine; Antihypertensive 

Introduction

The angiotensin I converting enzyme (ACE-EC 3.4.15.1) is a widespread zinc-metallopeptidase that converts angiotensin I (AI) to the vasoconstrictor octapeptide angiotensin II (AII). ACE also inactivates bradykinin, the vasodilator peptide opponent to AII (Michaud et al., 1997). AII plays a key role in the physiological up-regulation of blood pressure (BP, Waeber et al., 1995) either via direct interaction with [AT.sub.1]/[AT.sub.2] receptors in the cardiovascular system and CNS (Regitz-Zagrosek et al., 1995; Oparil et al., 2005), or by stimulation of aldosterone secretion, or by inhibition of prostaglandin synthesis (Zaman et al., 2002). ACE inhibitors and AT1 blockers reestablish BP towards basal level without many serious collateral effects (Oparil et al., 2005).

The important therapeutic role of ACE inhibitors in hypertension and congestive heart failure led to intensive search for natural molecules (Lacaille-Dubois et al., 2001) and synthesis of new enzyme inhibitors (Michaud et al., 1997; Choo et al., 2000). Either in vitro or in vivo screening methods for compounds endowed with ACE inhibitory activity have been proposed; results from the former procedure are faster but often are not confirmed in vivo (Wagner, 1993).

Brazilian folk medicine refers to Cecropia sp respiratory, cardiac and diuretic activities (Matos, 1989). In previous studies, we have shown that oral administration of the standardized aqueous extract (AE) of C. glaziovii leaves and its purified butanolic fraction (BuF) produced sustained and reversible hypotension in normotensive and hypertensive rats. Putatively, the effect was attributed to L-type calcium channel blockade (Lapa et al., 1999; Lima-Landman et al., 2006).

However, Castro Braga et al. (2000) and later Lacaille-Dubois et al. (2001) described in vitro reduction of ACE activity by the dicloromethane/methanol (1:1) extract of C. glaziovii. This effect was not confirmed in anesthetized rats injected with BuF (Lima-Landman et al., 2006), but because the enzyme activity was indirectly evaluated on the pressor responses to i.v. injected AI, further experiments were necessary to discard the putative ACE inhibition. The results now reported show that plasma ACE activity during acute or long term oral treatment with the standardized AE of C. glaziovii do not change and thus do not correlate to the hypotension simultaneously recorded. Comparatively, a direct correlation between hypotension and decreased ACE activity was observed when the same rats were treated with captopril, a known ACE inhibitor.

Material and methods

All the experimental protocols were approved by the Institutional Ethical Committee (Unifesp CEP 1233/00).

Plant standardized extract

The plant origin and preparation of the standardized AE were detailed elsewhere (Lima-Landman et al., 2006). In short, the plant was cultivated, the leaves were dried and ground. The 2% AE was prepared at 72 [degrees]C, filtered, concentrated under vacuum and freeze-dried. AE was standardized by its content in catechins (12%), procyanidins (19%) and flavonoids (19%) all of them chemically identified (Tanae et al., 2006). Captopril was purchased from Sigma (USA).

Direct measurement of blood pressure and heart rate from conscious rats

Female normotensive Wistar rats (200-300 g) anesthetized with sodium pentobarbitone (60 mg/kg, i.p.) were prepared for BP recording through a PE10 cannula implanted in a femoral artery and exteriorized at the dorsal neck skin. Heparine (10 UI/ml) was used to avoid clothing inside the cannula. After recovery from anesthesia, the rats were individually housed and observed for at least 24 h.

Mean arterial pressure (MAP) and heart rate (HR) from non-anesthetized animals were monitored with a Gould-Stathan pressure transducer (P23 ID, USA) connected to a polygraph. After a 10min of basal recording, either AE (1 g/kg), captopril (50 mg/kg), or vehicle (water--5ml/kg) were administered with an orogastric cannula and the effects on MAP and HR recorded after 30min, 1, 2, 4, 6, 12 and 24 h of treatment.

Indirect measurement of blood pressure from conscious rats

Systolic BP in non-anesthetized rats was measured by non-invasive tail-cuff plethysmography as described before (Lima-Landman et al., 2006). During the control period of water administration (2-4 weeks) and after drug treatment, BP was monitored once a week. Either the AE of C. glaziovii (0.5 g/kg), the vehicle (water--5 ml/kg) or captopril (50 mg/kg) were administered intragastrically bid at 9:00 am and 5:00 pm. BP measurement preceded the blood sampling and both were performed prior to the animal treatment unless otherwise stated.

Blood sampling

Once a week, about 0.5 ml of blood was taken from the tail artery with a heparinized syringe under light ether anesthesia. The plasma was separated by centrifugation (10 min, 2500 rpm, 10 [degrees]C) and stored at -20 [degrees]C until the moment of the enzymatic activity determination.

ACE activity determination

The AI converting enzyme (ACE) activity in rat plasma was determined using the indirect fluorometric method described by Santos et al. (1985). The method is based on the hydrolysis of Hypuril-L-Histidine-L-Leucine by plasma ACE producing the dipeptide L-Histidine-L-Leucine (His-Leu) which was quantitatively coupled to the fluorescent probe ortho-phtaldial-dehyde measured at [[lambda].sub.ex] = 360 nm; [[lambda].sub.em] = 500 nm. The ACE activity (nmol/ml/min) was determined by interpolation in a standard curve of His-Leu.

Statistics

The results were expressed as mean [+ or -] standard error of the mean and submitted to ANOVA followed by the Dunnet test, with p < 0.05.

Results

Mean arterial blood pressure (MAP), heart rate (HR) and plasmatic ACE activity in non-anesthetized normotensive rats after single oral administration of the aqueous extract (AE) of C. glaziovii

In normotensive rats, the MAP was 122.2 [+ or -] 1.2 mm Hg, the HR was 297.7 [+ or -] 5.5bpm and the plasma ACE activity was 32.9 [+ or -] 1.5 nmol/ml/min (n = 30).

These values were not significantly altered within 24 h of either the extract (AE 1g/kg, p.o., n = 8) or the vehicle treatment (Fig. 1). As expected, captopril (50 mg/kg, p.o., n = 14) significantly reduced the MAP by 7-16% from 30 min to 24 h afterwards. The HR increased by 10-20% during the same period. The ACE activity was reduced by 10% after 30 and 60min of captopril administration, recovering to basal levels within 12 h, and increasing to values 32% higher than the basal after 24h of administration (basal = 30.7 [+ or -] 2.6 nmol/ml/min) (Fig. 1).

Systolic blood pressure (BP) and plasmatic ACE activity in non-anesthetized normotensive rats after repeated treatment with the aqueous extract (AE) of C. glaziovii

The mean basal BP measured in the rat tail was 121.7 [+ or -] 0.5 mm Hg (n = 39) leveling through the 4 weeks of water administration. The mean plasmatic ACE activity considering all rats was steady at 37.6 [+ or -] 1.7 nmol/ml/min. ACE values dispersed from 18.3 nmol/ml/min to 51.8 nmol/ml/min among the same animals, although remaining constant for each rat (Fig. 2). Taking into account that the ACE dispersion might obscure drug effects, the rats were grouped in three groups with significantly different ACE plasmatic activity: high-ACE = 48.8 [+ or -] 2.6 nmol/ml/min, n = 10; intermediate ACE activity (i-ACE = 39.4 [+ or -] 1.4 nmol/ml/min, n = 19) and low ACE activity (low-ACE = 23.5 [+ or -] 0.6 nmol/ml/min, n = 10) (Fig. 2). Nine rats of the i-ACE group were taken for control during the following experiments.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The basal systolic BP was not different in animals with high-ACE, i-ACE and low-ACE (BP = 122.5 [+ or -] 1.9 mm Hg; 124.0 [+ or -] 1.3 mm Hg and 124.5 [+ or -] 1.9 mm Hg, respectively). The repeated treatment with the AE (0.5 g/kg/bid, p.o.) significantly reduced the BP from the 14th day throughout treatment in all groups. Within 30 days, BP was reduced to 106.9 [+ or -] 1.9 mm Hg; 108.0 [+ or -] 1.7 mm Hg and 102.8 [+ or -] 1.2 mm Hg in the rats with high-ACE, i-ACE and low-ACE, respectively (Fig. 3A). The hypotension observed was not statistically different among the groups. BP in control group receiving water did not change (120.6 [+ or -] 1.8 mm Hg). Likewise, the ACE activity did not significantly change in any group after 30 days treatment with AE. The enzyme activities were 45.6 [+ or -] 2.7; 37.9 [+ or -] 0.1 and 22.0 [+ or -] 0.5 nmol/ml/min in high-ACE, i-ACE and low-ACE rats, respectively (Fig. 3B).

During the washout period, when all animals were treated with the vehicle (water 0.5 ml/kg/bid, p.o.), the BP was restored to values not different from control (123.3 [+ or -] 1.9, 121.5 [+ or -] 1.3 and 121.9 [+ or -] 0.9 mm Hg) in high-ACE; i-ACE and low-ACE rats, respectively, without changes in ACE activities (50.0 [+ or -] 1.55, 40.8 [+ or -] 1.9 and 24.2 [+ or -] 0.2 nmol/ml/min) in the same groups (Fig. 3).

Once BP was restored to control values, the rats were treated with captopril (50 mg/kg/bid, p.o.), for 56 days. Within a week, the BP was reduced to 108.5 [+ or -] 0.8 and 107.5 [+ or -] 1.3 mm Hg in animals with i-ACE and low-ACE, respectively. In high-ACE rats, BP was significantly reduced only after 14 days of treatment with captopril (103.0 [+ or -] 1.1 mm Hg). Comparatively, BP in the vehicle treated group did not change (123.3 [+ or -] 1.4 mm Hg, Fig. 3).

[FIGURE 3 OMITTED]

In view of the dual ACE response to captopril administration, i.e., initial inhibition followed by up-regulation (Fig. 1), the weekly blood sampling was alternately performed 1 and 12 h after the first daily dose. The protocol for BP measurement was unchanged.

The plasmatic ACE activity measured 12h after the drug administration (i.e., immediately before the second daily dose of captopril) was 123% (108.7 [+ or -] 4.9 nmol/ml/min), 142% (95.3 [+ or -] 2.3 nmol/ml/min) and 114% (50.2 [+ or -] 1.5 nmol/ml/min) above basal levels in animals with high-ACE, i-ACE and low-ACE, respectively. One hour after captopril administration, however, the ACE activity in the same groups was 51%, 55% and 59% lower than that detected 12h after treatment, respectively (Fig. 4). Comparatively, in the vehicle treated rats ACE activity was unchanged (39.4 [+ or -] 1.4 nmol/ml/min). Switching the captopril treatment to the vehicle administration (washout) caused the return of the BP and ACE activity to control values (Fig. 3).

Once re-established the control condition after washout, a second treatment with AE (0.5 g/kg/bid, p.o.) reduced BP by 10% in all groups but the plasma ACE activity was unchanged (Fig. 3), confirming the previous results.

[FIGURE 4 OMITTED]

Discussion

The present paper aimed to evaluate the effect of the standardized AE of Cecropia glaziovii on plasma ACE activity during the hypotension produced by the extract in normotensive rats.

The ACE inhibitors are among the best antihypertensive medicines available. Their effect is explained by diminished AII production and decrease of its endocrine, autocrine and paracrine actions. Decreased aldosterone and increased prostaglandin secretion are other relevant effects of these compounds (Oparil et al., 2005).

In vitro screening of medicinal plants is a fast method often used to search for new ACE inhibitory compounds (Wagner, 1993; Adsersen and Adsersen, 1997; Nyman et al., 1998; Duncan et al., 1999; Somanadhan et al., 1999). Many plant derived compounds inhibit in vitro ACE activity including proanthocyanidins (Inokuchi et al., 1986; Wagner, 1993; Wagner and Elbl, 1992), tannins (Ueno et al., 1988), phenylpropanes (Yamadaki and Shimoyama, 1993), peptides (Wagner, 1993; Kinoshita et al., 1993), cyclooctapeptides (Li et al., 2004), xanthones (Chen and Lin, 1992), fatty acids (Morota et al., 1987), terpenoids (Morigiwa et al., 1986); alkaloids (Ogino et al., 1988) and flavonoids (Wagner, 1993; Hansen et al., 1996; Lacaille-Dubois et al., 2001).

In vivo studies are few. For example, animals treated with quercetin, a common plant constituent, administered i.v. or p.o., presented lower ACE activity than those receiving saline by the same route (Hackl et al., 2002).

Castro Braga et al. (2000) described in vitro ACE inhibition (20%) by the dichloromethane/methanol extract of Cecropia glaziovii. The effect was not attributed to specific components of the plant extract. The flavonoids vitexin and isovitexin were isolated from the plant (Della Monache et al., 1988; Lacaille-Dubois et al., 2001; Tanae et al., 2006) and shown to inhibit in vitro ACE activity by 20% and 45%, respectively (Lacaille-Dubois et al., 2001), but this effect was not confirmed by others (Bormann and Melzig, 2000).

The hypotension induced by Cecropia extracts has been studied before. Acute oral treatment with AE did not alter BP within the first week of oral treatment (Lapa et al., 1999; Lima-Landman et al., 2006).

These findings were accurately confirmed in the present work by direct recording BP during 24 h after oral administration. Comparatively, captopril (50 mg/kg, p.o.) decreased BP significantly after 30 min, increased the HR, probably by a reflex mechanism, and significantly reduced the ACE activity within the first hour after administration. The enzyme activity was recovered within 6-12 h and it was clearly 32% above the basal level after 24 h treatment. This response confirmed the up-regulation of ACE described by other authors in animals (Kokubu et al., 1980; Forslund et al., 1981) and in humans (Rosenthal et al., 1982; Lechi et al., 1983).

The latency for the hypotensive effect induced by Cecropia may depend on late activation of modulatory mechanisms, as observed in the CNS (Rocha et al., 2002), or may be due to less bioavailability of the substances responsible for the hypotensive effect. A short delay for the anti-acid effect after AE administration into the duodenal lumen was described by Souccar et al. (2006). Therefore, it seems that different compounds and pathways are probably involved on Cecropia effects. More evidences are needed to clarify the missing mechanisms of these actions.

The present experiments focused on BP effects and ACE activity. In any case the changes in BP produced by AE were correlated to changes in ACE activity. It was clear that the values for ACE activity varied by three fold in random selected rats but it was constant for each animal. In fact, about 50% of the animals presented intermediate values for ACE, 25% presented two times higher ACE activity and 25% had a forth of the high ACE activity. This natural dispersion of ACE activity could mask drug effects but AE treatment did not alter the enzyme activity in any of the experimental groups. This result corroborate the previous finding that the hypertension produced by AI was unchanged after treatment with Cecropia extracts (Lima-Landman et al., 2006). They also exclude the involvement of the renin-angiotensin system on the hypotensive effect of Cecropia glaziovii, opposite to captopril, the positive control. Finally, the reversal of BP to basal levels after drug washout reinforces the potential safety of the plant extract.

In conclusion, repeated treatment with the AE of Cecropia glaziovii induced hypotension in normotensive rats. This hypotensive effect was not related to inhibition of ACE plasma activity. So far, the blockade of [Ca.sup.2+] influx in vascular smooth muscles remains as the putative mechanism of the plant hypotensive effect (Lapa et al., 1999).

Acknowledgments

The authors express their gratitude to Drs. Adriana Carmona and Dulce Casarini for helpful comments and for some drug supply. Thanks is also due to Joao Francisco R. dos Santos, Celso M. das Dores and Alex S. de Oliveira for the technical support during the experiments.

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M.F.M.L. Ninahuaman, C. Souccar, A.J. Lapa, M.T.R. Lima-Landman*

Universidade Federal de Sao Paulo, Department of Pharmacology, Natural Products Section, 04044-020 Sao Paulo, SP, Brazil

Received 23 June 2006; accepted 7 November 2006

*Corresponding author. Tel.: + 55 11 5576 4447; fax: + 55 11 5576 4499.

E-mail address: [e-mail] (M.T.R. Lima-Landman).