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Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles.

Abstract

The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to l000 [mu]g/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 [mu]M) or KCI (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 [mu]M), an antagonist of [[alpha].sub.1]-adrenoceptors, atropine (0.1 [mu]M), an antagonist of muscarinic receptors, and ritanserin (50 nM), a [5-HT.sub.2A] antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD.

We also investigated the action of SdD (10-1000 [mu]g/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 [mu]g/ml) the contraction induced by carbachol (1 [mu]M) was increased, whereas that by KCI (60 mM) or capsaicin (100 nM) were unchanged.

The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to [[alpha].sub.1], M, [5-HT.sub.2A] and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles.

[C] 2009 Elsevier GmbH. All rights reserved.

Keywords: Croton lechleri; Euphorbiaceae; Constriction; Caudal artery; Gastric fundus; Rat

Introduction

Croton lechleri L. (Euphorbiaceae) known as Sangre de Drago (SdD) or dragon's blood is one of the most widely used medicinal plants of the South American tropics. The blood-red tree sap is traditionally used to treat many illnesses (Jones 2003) and recently, this sap has become available in many countries as a dietary supplement. SdD is a crude drug which is used topically in the treatment of wound healing and orally, in a dilute form, mainly for stomach ulcers, ulcerative colitis, dysentery and diarrhoea (Miller et al. 2000). The combination of antimicrobial, antioxidant, antiviral and cicatrizing properties makes this red sap a complex herbal remedy of pharmacological interest (Williams 2001; Risco et al. 2003). The chemical constituents of the sap from C, lechleri have already been thoroughly investigated, for which reason we did not look further into this matter. Authors have shown that more than 90% dry weight of the sap consists of mixtures of proanthocyanidins such as catechin, epicatechin, gallocatechin, epigallocatechin and dimeric procyani-dins (Cai et al. 1991; Phillipson 1995). Other minor components are clerodane diterpenoids korberin A and B (Cai et al. 1993a, b), 1,3,5-trimethoxybenzene and 2,4,6-trimethoxyphenol, lignan 3', 4-O-dimethylcedrusin, and alkaloid taspine (Phillipson 1995; Vaisberg et al. 1989; Pieters et al. 1993). Recently, authors have identified other minor components such as blumenols B and C, 4,5-dihydrobIumenol A and floribundic acid glucoside (De Marino et al. 2008). In order to have the best definition of the tested product, prior to the activity testing, SdD samples were characterised using different chromatographic and spectroscopic methods.

During the research, we examined the activity of the dried red sap from C. lechteri (SdD) on both rat caudal artery and gastric fundus, two experimental models of smooth muscles, to focus on the vascular and gastric effects of this traditional remedy as no data is available on this matter in scientific literature.

Materials and methods

Plant material, chemicals and instrumentation

The red sap was obtained in the traditional way, carving the bark of trees growing in the province of Napo, Ecuador. The voucher code No: SdD 005 for the crude drug was deposited in the Department of Pharmaceutical Sciences of Padua University.

The sap was freeze-dried in a lyophilizer and stored in the dark at -20 [degrees]C.

Chemicals, reagents and solvents, were purchased from Aldrich or Fluka and used without further purification. When necessary the solvents where HPLC grade. (1) H and (13) C NMR spectra were recorded on a Bruker AMX300 spectrometer in deuterated methyl sulfoxide (DMSO-d6) from Cambridge Laboratory. Chromatography and flash chromatography were performed using silica gel Merck 60 (70-230 mesh) and Merck 60 (40-63 pm) respectively. TLC plates were also from Merck: Merck 60 F-254, 0.25 mm precoated plates. Melting points were determined using a Gallenkamp apparatus in capillary tubes. Elemental analysis (C, H, N) was performed by the Microanalytical Laboratory of the Department of Pharmaceutical Sciences of the University of Padova on a Carlo Erba 1016 elemental analyzer, and the observed values are within 0.40% of the calculated values. Exact masses (HRMS) were obtained using a Mariner API-TOF (Perseptive Biosys-tems Inc., Framingham MA 01701), and found and calculated m/z values are given. Analytical HPLC was carried out on a Varian ProStar system with a Star Chromatography Workstation (version 5.5.1; Varian, Inc., Walnut Creek, CA) at a flow rate of 1 ml/min using Varian ProStar 210 pumps, with a 10 [mu]l loop and with a ProStar 325 UV/V detector, signals were monitored at 254 nm. The separations were performed on a Zorbax Agilent Eclipse XDB C-8 (150 x 4.6 mm), 5 [mu]m column.

Characterisation of sap from Croton lechleri

Before lyophilisation, pH and density of the sap were determined. Dry residue weight after lyophilisation was also measured. The presence of polyphenols and of the alkaloid taspine was investigated initially by TLC (Risco et al. 2003; Cai et al. 1991; Perdue et al. 1979).

In order to have a chromatographic fingerprinting of red sap from C. lechleri that was in our hands we performed an HPLC of the crude extract, recording the UV spectra of the single peaks (Diode Array Detector). Chromatography was carried out in a gradient system with a flow rate of 1.0 ml/min. The mobile phase consisted of eluent A (5% acetic acid in water) and eluent B (5% acetic acid in methanol). The starting eluent was 95% A and 5% B, after 5 min the proportion of eluent B was increased linearly to 70% in 15.0 min and kept at this composition for 5.0 min, then B was increased linearly up to 90% in 10.0 min and kept at 90% for additional 5.0 min and then returned to initial composition of eluent A (95%) and B (5%) in 3.0 min followed by another 5.0 min to re-equilibrate the column. The detector wavelength was set at 254 nm for runs at fixed wavelength to determine taspine while it was set at 220 nm to obtain a fingerprint of fresh sap (Fig. 1).

[FIGURE 1 OMITTED]

Taspine was then isolated by preparative column chromatography on alumina (dichoromethane:metha-nol 20:1) and the purity determined by HPLC (area %) while the structure was confirmed by (l) H-NMR, mass spectral data and elemental analysis. The NMR data were compared with the literature (Kelly and Xie 1998).

In vitro pharmacology

The present study conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No 85-23, revised 1985). Adult male rats six-months old (Wistar, 300-400 g) were anesthetised through inhalation of methoxyflurane and then sacrificed; the stomach and the caudal artery were removed and rinsed with Krebs solution. The fundus region of the stomach was excised; strips of longitudinal muscle layer (3 mm wide and 1 cm length) were mounted in organ baths under a resting tension of 9.8 mN. Also the proximal 2 cm of the caudalartery was cleaned of adhering tissue and the arterial rings 3 mm long were placed in a tissue bath under a resting tension of 19.6 mN. Extreme care was taken not to damage the endothelium. Otherwise, in some experiments, the endothelial cell layer was removed by rubbing the luminal side of the vessel with an L-shaped stainless steel wire. The tissue bath was filled with Krebs-Ringer solution at 37 [degrees]C, with the following composition (mM): NaCl 118.3, KC1 4.7, [CaCl.sub.2] [2H.sub.2.O] 2.5, [MgSO.sub.4] [7H.sub.2]O 1.2, [KH.sub.2][PO.sub.4] 1.2, [NaHCO.sub.3] 25, D-Glucose 11.1, was bubbled with 95% [O.sub.2] and 5% [CO.sub.2] ([pO.sub.2] = 345 [+.or.-]8 mmHg), pH = 7.2. Tissues were allowed to equilibrate for 1 h before viability was assessed with standard start procedures. The isometric tension was recorded by means of a high-sensitivity transducer connected to a chart recorder (Ugo Basile, type DYO, and Unirecord System, Model 7050, Comerio (VA), Italy).

In experiments carried out on slightly depolarised tissue, the Krebs-Ringer solution was modified in order to counterbalance the increased concentration of KC1 (30 mM) by an equal reduction of NaCl.

Groups of rats were treated with reserpine 2.5mg/kg i.p., 48 and 24 h before sacrifice, to deplete them of endogenous reserves of catecholamines (Crivellato et al. 2006). Arterial tissues from reserpine-treated rats were tested with tyramine in order to evaluate endogenous levels of catecholamines: preparations responding to tyramine with vasoconstriction were not considered.

Drugs

Ritanserin and prazosin were prepared in dimethyl-sulfoxide. All other drugs were dissolved in saline. The concentration of organic solvent added never exceeded 0.2% (V/V) of the organ bath; in this condition, the solvent did not modify the contractility of tissues. Compounds were obtained from Sigma-RBI (USA). All reagents were of analytical grade. The purity of all compounds was > 98%.

Statistical analysis

All values presented are means [+.or.-] S.E.M. of 6-10 experiments. Changes in vascular tension are expressed as percentage of the contraction induced by 10 [mu]M phenylephrine taken as 100%, while variations of gastric tension are the percent of the effect induced by the agonist (carbachol or KCl). All concentrations expressed indicate the final concentration in the tissue bath. [E.sub.max] is the maximum response evoked by an agonist, while [EC.sub.50] is half the maximum effective concentration.

Sigmoid curve fitting performed by the GraphPadPrism program (GraphPad Software, San Diego, CA, USA). Based on the principal equation for a sigmoidal curve, the program makes itinerated computations to derive a best fit, based upon the actual experimental values.

For statistical comparisons between treatment and control data, student's two-tail paired t-test was used; the difference between mean group data was significant at p < 0.05.

Results

The pH of fresh red sap of C. lechleri (SdD) was 3.9 and the density was 1.09 g/ml according to literaturedata (Risco et al. 2003). SdD (l0 [mu]l/ml) per se slightly changed basal contractile tension in caudal arteries (Fig. 2), but not in gastric fundus strips (data not shown).

[FIGURE 2 OMITTED]

To permit the conservation of the sap and to standardize experiments, we freeze-dried the fresh sap obtaining a crystal brown powder with a dry residue of 24%. The dried sap, tested from 10 to 1000 [mu]g/ml, per se increased basal arterial tension; each concentration caused vasoconstriction related to the amount of SdD added to the organ bath (Figs. 3 A and B). Also the SdD cumulative concentration-curve effect was obtained starting from 10[mu]g/ml and reaching the maximum at 500 [mu]g/ml (Figs. 4 A and B). The calculated EC50 was about 235[mu]g/ml.

[FIGURE 3 OMITTED]

We observed that 1000 [mu]g/ml dried sap always gave lather in the organ bath and, for this reason, we did not use higher concentrations. Vasoconstriction induced by SdD was rapidly reversible after a washout. We also evaluated the role of endothelmm-derived relaxing factor NO by using caudal arteries deprived of endothelium; in this condition, the vasoconstrictioninduced by SdD, either single or cumulative concentrations, was not modified (data not shown).

In a further study, SdD was applied to tissues pre-constricted either by 0.1 [mu]M phenylephrine or 30 mM KC1. In both experimental conditions, SdD (10-1000 [mu]g/ml) caused higher concentration-dependent vasoconstriction that was more evident at 100 [mu]g/ml (Fig. 4 B).

[FIGURE 4 OMITTED]

We also tested SdD in caudal arteries from rats that were treated in vivo with reserpine, to deplete tissues of endogenous catecholamines (Crivellato et al. 2006). In this experimental condition, vasoconstriction induced by SdD showed similar magnitude and potency compared to that obtained in tissues from untreated rats, both in basal and in pre-constricted (0.1 [mu]M phenylephrine) tissues (data not shown).

The involvement of [alpha]-adrenoceptors was tested using arterial tissues pre-incubated for 30min with 0.l[mu].M prazosin, a selective inhibitor of [[alpha].sub.1],-receptor (Pares-Hipolito et al. 2006), Prazosin alone did not influence contractile tension nor the vasoconstriction induced by SdD (Fig. 5 A). Atropine (0.1 [mu]M, 30 min), an antagonist of muscarinic receptors (Hulme et al. 1990), alone did not change basal tension or modify the effect induced by SdD (Fig. 5 B). The role of the [5-HT.sub.2A] receptor subtype was also examined by incubating arterial tissue with ritanserin (50 nM, 30 min), a well-known [5-HT.sub.2A] antagonist (Hoyer and Martin 1997). Ritanserin alone did not change the arterial tension nor the concentration-effect curve of SdD (Fig. 5 C).

[FIGURE 5 OMITTED]

Nifedipine (50 nM, 30 min), an inhibitor of L-type calcium channels (God fraind 1987), weakly decreased basal arterial contractile tension but pre-treatment with this compound did not modify the vasoconstriction induced by SdD (Fig. 6 A). Capsaicin is a pungent compound from Capsicum spp. (Solanaceae) used as a pharmacological tool to investigate the role of vanilloid receptors (Szallasi and Blumberg 1999). Capsaicin (0.1 [mu]M, 30 mm)per se slightly increased the contractile tension without affecting the vasoconstriction caused by SdD (Fig. 6 B).

[FIGURE 6 OMITTED]

In studies on rat gastric fundus in vitro SdD, tested from 10 to 1000 [mu]g/ml, per se increased smooth muscle tension (Fig. 7). In order to go into the action of SdD al gastric level, we evaluated its influence on the increased tension induced by carbachol, a well known muscarinic agent and by K.C1, a depolarizing agent. The contraction caused by 1 [mu]M carbachol increased by pre-treatment with 100 [mu]g/ml SdD (Fig. 8 A), whereas the response to 60 mM KC1 remained unchanged (Fig. 8 B). We also tested capsaicin on rat gastric fundus; the alkaloid (100 nM) per se increased smooth muscle tension of 10[+or_]3.0% (p<0.05). The preincubation with 100 [mu]g/ml SdD did not change the contraction caused by capsaicin (data not shown).

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

Discussion

The main finding from this study is that the sap of C. lechleri increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles, indicating the presence of active compound(s) in the sap.

How does the sap increase the contractile tension? We studied vasoconstriction by SdD using various types of pharmacological tools acting at receptor subtypes or ionic channels regulating vascular tension. Atropine, a well known competitive muscarinic antagonist, did not influence vasoconstriction induced by SdD, suggesting that muscarinic activation is not responsible for the effect of SdD. We also excluded the role of [[alpha].sub.1] and [5-HT.sub.2A] receptors by using prazosin, a well known selective antagonist of a [[alpha].sub.1] adrenergic receptors, and ritanserin, a [5-HT.sub.2A] selective antagonist, as neither inhibitor influenced vasoconstriction caused by SdD.

It is well known from the literature that compounds which increase intracellular calcium level produce vasoconstriction in vessels and various vasoconstricting agents act, either directly or indirectly, in this way. The pre-treatment with nifedipine, one of the first known inhibitors of L-type calcium channels, did not inhibit the contractile effect by SdD excluding a direct action on these channels by the sap. Finally, we studied the influence of capsaicin, an alkaloid agonist of vanilloid receptors (VRs) mainly present in sensory neurons but also in nonneuronal tissues (Szallasi and Blumberg 1999), and recently discovered in vascular smooth muscle too (Kark et al. 2008). The pre-treatment with capsaicin did not influence vasoconstriction by SdD suggesting that these receptors are also not involved in the action of the sap.

The data indicates that vasoconstriction induced by the SdD is not related to activation of M, [[alpha].sub.1], [5-HT.sub.2A] and vanilloid receptors or the L-type calcium channels. Therefore, the underlying mechanism(s) responsible for the vasoconstriction by SdD requires further study.

SdD has also been shown to have wound healing properties, which may result from a number of factors including the strong antimicrobial activity of polyphenols and the anti-inflammatory and radical scavenging properties of the proantocyanidins (Phillipson 1995). From our in vitro experiments we suggest that vasoconstriction could also be an important factor in tissue repair.

SdD has been used traditionally in the treatment of gastrointestinal diseases (Jones 2003). Our experiments showed that SdD increased contractile tension of gastric fundus and also increased the contraction induced by carbachol, whereas it did not change contraction induced by depolarization or capsaicin. These results could support the traditional use of SdD on gastric ailments.

Overall, the data shows that the red sap obtained from C. lechleri could be a precious source of substance(s) acting on smooth muscle, especially vascular muscle. Thus, the present research is the first step in our study of SdD. The second will be the identification of the agent(s) acting on smooth muscles, which could be useful to increase the knowledge of C. lechleri, an under investigated medicinal plant, and possibly to develop new pharmacological agents affecting smooth muscle activity.

Acknowledgements

The authors wish to thank "II Crisolito"s.r.l. (Spresiano, Treviso Italy) and Padri Giuseppini for providing the plant materials. The authors are grateful to Elena Baldon for performing some experiments while preparing her degree thesis. We are also grateful to Dr. L. Solda and Mr. E. Secchi for technical and Mr. S. Lovison for software assistance, Dr. R. Sato and Mrs. F. Chinaglia for reference assistance and Sara Eames for the English text revision.

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G. Froldia (a), *, G. Zagotto (b), R. Filippini (c), M. Montopoli (a), P. Dorigo (a), L. Caparrotta (a)

(a) Department of Pharmacology and Anaesthesiology, University of Padova, Largo E. Meneghetti 2, 35131 Padua, Italy

(b) Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padua, Italy

(c) Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padua, Italy

*Corresponding author. Tel.: + 39498275092; fax: + 39498275093. E-mail address: g.froldi@unipd.it (G. Froldi).

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doi:10.1016/j.phymed. 2009.02.003
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Author:Froldia, G.; Zagotto, G.; Filippini, R.; Montopoli, M.; Dorigo, P.; Caparrotta, L.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUIT
Date:Aug 1, 2009
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