2005-ticli rosmarinic acid

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Rosmarinic acid, a new snake venom phospholipase A2 inhibitor

from Cordia verbenacea (Boraginaceae): antiserum

action potentiation and molecular interaction

Fabio K. Ticlia, Lorane I.S. Hagea, Rafael S. Cambraiab, Paulo S. Pereirab,Angelo J. Magroc, Marcos R.M. Fontesc, Rodrigo G. Stabelid, Jose R. Giglioe,

Suzelei C. Francab, Andreimar M. Soaresa,*, Suely V. Sampaioa

a Departamento de Ana lises Clı nicas, Toxicolo gicas e Bromatolo gicas, FCFRP, Universidade de Sa o Paulo,USP, Ribeira o Preto-SP, Brazil

bUnidade de Biotecnologia, Universidade de Ribeirao Preto, UNAERP, Ribeirao Preto-SP, Brazilc Departamento de Fı sica e Biofı sica, IB, Universidade Estadual Paulista, UNESP, Botucatu-SP, Brazild Laborato rio de Bioquı mica do Instituto de Pesquisas em Patologias Tropicais (IPEPATRO), FioCruz,

UNIR, Porto Velho-RO, Brazile Departamento de Bioquı mica e Imunologia, FMRP, Universidade de Sa o Paulo, USP, Ribeira o Preto-SP, Brazil

Received 3 February 2005; revised 27 April 2005; accepted 28 April 2005

Available online 29 June 2005

Abstract

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Themethanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu

snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active

component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as

rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2- O-cafeoil-3-

(3,4-di-hydroxy-phenyl)- R-lactic acid]. This is the first report of RA in the species C. verbenacea (‘baleeira’, ‘whaler’)

and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the

edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit

the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2

from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the

myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between

the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine

polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in

Toxicon 46 (2005) 318–327

www.elsevier.com/locate/toxicon

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2005.04.023

Abbreviations Cv-ME, Cordia verbenacea methanolic extract; Cv-RA, rosmarinic acid from Cordia verbenacea; PLA2, phospholipase A2;

PLIs, phospholipase A2 inhibitors; BthTX-I, B. jararacussu bothropstoxin-I; BthTX-II, B. jararacussu bothropstoxin-II; BthA-I-PLA2, B.

jararacussu acidic phospholipase A2; COSY, COrrelation SpectroscopY; HMQC, heteronuclear multiple quantum coherence; HMBC,

heteronuclear multiple bond coherence; CD, circular dichroism.* Corresponding author. Tel.:C55 16 602 4714; fax: C55 16 633 1936.

E-mail addresses: [email protected] (A.M. Soares), [email protected] (S.V. Sampaio).

http://www.elsevier.com/locate/toxicon

http://www.elsevier.com/locate/toxicon



experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either

in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is

proposed.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Cordia verbenacea; Rosmarinic acid; Anti-inflammatory; Antimyotoxic; Antiophidian; Phospholipase A2 inhibitor; Bothrops

jararacussu; Snake venom

1. Introduction

Plants have often been used by humans, sometimes

successfully, against numerous diseases caused by different

pathological agents. Pharmacological studies have demon-

strated that the extracts and fractions from some of these

plants used in traditional medicine possess anti-inflamma-

tory, antiviral and antiophidian properties (Phillipson and

Anderson, 1989; Martz, 1992; Mors et al., 2000). The

antiophidian activity of several plant species in general use

in some Brazilian communities has been investigatedscientifically (Mors et al., 2000; Batina et al., 2000; Borges

et al., 2000, 2001; Biondo et al., 2003, 2004; Januario et al.,

2004; Veronese et al., 2005; Esmeraldino and Sampaio, in

press; da Silva et al., in press; Oliveira et al., 2005).

Snake venoms are complex mixtures of proteins

including phospholipases A2, myotoxins, hemorrhagic

metalloproteases and other proteolytic enzymes, cytotoxins,

cardiotoxins and others. The pathophysiology of snake

envenomation involves a complex series of events that

depend on the combined action of these venom components

(Gutierrez, 2002). Phospholipases A2 (PLA2; EC 3.1.1.4)

are abundant in snake venoms. Besides playing a digestiverole in phospholipid hydrolysis, they may also exert a wide

variety of pharmacological activities such as neurotoxicity,

myotoxicity, edema-inducing activity and others (Gutierrez

and Lomonte, 1995; Soares et al., 2004b). Local edema, a

typical manifestation of Bothrops envenomation, usually in

addition to pain, is due to the action of the venom upon

mastocytes, kininogens and phospholipids, culminating with

release of endogenous mediators (Teixeira et al., 2003).

The hydroalcoholic extract from Cordia verbenacea

(‘baleeira’, ‘whaler’) has been used by Brazilian folk as

cicatrizant and anti-inflammatory (Sertie et al., 1988). We

report now, for the first time, the anti-inflammatory and

antimyotoxic activity of the extract from C. verbenacea andits

active principle, rosmarinic acid, against these effects induced

by Bothropsjararacussu snake venom andby its main isolated

phospholipases A2. A possible model for the interaction of

rosmarinic acid with Lys49-PLA2 BthTX-I is proposed.

2. Material and methods

2.1. Materials

The leaves from C. verbenacea were collected during

the blooming period in the Campus of the University of

Ribeirao Preto (UNAERP). A voucher specimen (No. 259)

identified by specialist Prof. Dr Lin Chau Ming (Departa-

mento de Botanica, UNESP, Botucatu, SP, Brazil) has been

preserved in the Unidade de Biotecnologia Herbarium,

UNAERP. B. jararacussu venom was purchasedfrom Sandrin

Bioagents serpentarium, Batatais, SP. B. jararacussu PLA2s

were isolated on Sephadex G-75 followed by cation-exchange

chromatography as previously described (Andriao-Escarso

et al., 2000, 2002). PLA2 homogeneity was assessed by native

and SDS-PAGE and reverse-phase HPLC.

2.2. Preparation of plant extract

After identification, the leaves were dried in a stove with

circulating air at 40 8C. They were then grounded (375 g)

and macerated with chloroform three times during three

days, then with methanol, followed by filtration and

evaporation of the methanol in a rotary evaporator where-

from the dried methanolic extract (Cv-ME) was obtained.

2.3. Purification and identification of rosmarinic acid

A preliminary Sephadex LH-20 column was used for thefirst fractionation of Cv-ME, using 300 mL of methanol as

mobile phase for elution. The resulting fractions, after

drying, were analyzed by thin-layer chromatography and

revealed with vanillin sulfuric acid reagent. Fraction 3 was

then applied on a HPLC semipreparative Supelcosil C18

column, using a concentration gradient of methanol:water at

a flow rate of 2 mL/min. Seven new fractions were so

obtained, which were assayed for edema inhibition, from

which rosmarinic acid (Cv-RA) was fraction 6, as identified

by NMR analysis. NMR spectra were recorded with a

Brucker DPX-300 spectrophotometer, operating at 300 mHz

for1

H and 75 mHz for13

C. For that, 15 mg samples wereused, dissolved in dimethyl-d 6-sulfoxide (Aldrich).

2.4. Edema-inducing activity

Edema was induced by i.d. injection, in the right foot pad

of male Swiss mice (18–22 g), of B. jararacussu venom

(25 mg) and its purified PLA2s (50 mg). Inhibition studies

were performed by incubating venom or PLA2 with

Cv-ME/Cv-RA. Control groups were injected with 50 mL

of phosphate-buffered saline (PBS, pH 7.2) alone, or

Cv-ME/Cv-RA alone. The progression of edema was

evaluated with a low pressure pachymeter (Mitutoyo,

F.K. Ticli et al. / Toxicon 46 (2005) 318–327 319



Japan) at various time intervals after injection (Soares et al.,

2000).

2.5. Enzymatic activities

PLA2 activity was determined in a gel plate containingegg yolk (6 egg yolks/L), CaCl2 (0.56 g/L) and agar (20 g/L)

, according to Gutierrez et al. (1988). Crude venom (1 mg),

BthTX-II (1 mg) and BthA-I-PLA2 (1 mg) were inoculated

and the diameter (mm) of the resulting halos were measured

after 2 h. Anticoagulant activity (Alvarado and Gutierrez,

1988) was evaluated using human plasma (200 mL), CaCl2(0.25 mM) and BthTX-II (0.5 mg). The plasma was

primarily equilibrated at 37 8C in a water bath with or

without Cv-RA/Cv-ME. BthTX-II was then added and, after

10 min, the CaCl2 solution (25 mL). Plasma which did not

clot after 45 min was considered incoagulable. The control

tube received PBS replacing BthTX-II, where the plasma

should clot within 3–6 min.

2.6. Myotoxic activity

Swiss male mice (18–22 g) were injected intramuscu-larly in the right gastrocnemius muscle with solutions

containing doses of 25 mg/50 mL of Bothrops venoms or

toxins. The mixtures of venom or toxin/Cv-RA (1:1 and

1:10, w/w) were then evaluated. Controls received PBS or

inhibitor alone. Mice were bled from the tail 3 h after

injections and blood was collected into heparinized capillary

tubes. Plasma creatine kinase activity was determined using

the Kit 47-UV (Sigma Chemical Co.) (Soares et al., 2000).

Activity was expressed in units/L, one unit corresponding to

the production of one micromole of NADH per min at 30 8C.

Fig. 1. Purification of Cv-RA from Cordia verbenacea methanolic extract. (A) Fractionation on Supelcosil C18, by HPLC, of fraction F3

from the Sephadex LH-20. (B) TLC of fraction F3-CL6 (rosmarinic acid). Mobile fase:ethyl acetate:formic acid:acetic acid:water

(100:11:11:26, v/v). Staining: NP/PEG, under UV. (C) Assay for purity of rosmarinic acid by HPLC-C18.




Fig. 2. Rosmarinic acid from Cordia verbenacea methanolic extract. (A) Molecular structure of Cv-RA from resonance studies (NMR 1H and

NMR 13C). (B) 3D-molecular structure of Cv-RA.

Fig. 3. Inhibition of the edema-inducing activity by rosmarinic acid. (A) Effect of Cv-ME (1:10, w/w) on the edema induced by B. jararacussu

crude venom, Lys49-BthTX-I and Asp49-BthTX-II. (B) Effect of Cv-RA (1:3.5, w/w) on the edema induced by the crude venom, BthTX-I and

BthTX-II. Results are expressed by the meanG

SD (nZ

6). Means are statistically significantly different from the control means.




2.7. Potentiation of anti-bothropic serum action

Cv-RA was added to polyvalent antivenom (Instituto

Butantan-SP, Brasil) at an ED50 (effective dose to neutralize

50% of myotoxicity as defined by levels of creatine kinase

in plasma after 3 h post-injection), incubated with

B. jararacussu venom or isolated PLA2s in a final volumeof 50 mL for 30 min at 37 8C and injected intramuscularly

in mice as previously described (Lizano et al., 2003).

2.8. Circular dichroism of Cv-RA

Far UV circular dichroism spectra (190–250 nm) were

measured with a JASCO 810 (JASCO, Inc., Tokyo, Japan)

using 1 mm path length cuvettes and protein concentrations

of 150 mg/mL for both the target myotoxic PLA2s and

450 mg/mL for Cv-RA. In the case of mixtures, the total

protein concentration was 150 mg/mL. In all cases, a total of

10 spectra were collected, averaged and corrected by

subtraction of a buffer blank.

2.9. Molecular modeling

The molecular model of the monomeric BthTX-I

(da Silva-Giotto et al., 1998) complexed with rosmarinic

acid was elaborated using the program O (Jones et al.,

1990). The complex was refined and its energy was

minimized using the program CNS (Brunger et al., 1998).

2.10. Statistical analysis

Results are presented as the mean valueGSD obtained

with the indicated number of tested animals. The statistical

significance of differences between groups was evaluated

Fig. 4. Inhibition of the PLA2 activity by rosmarinic acid. Effect of Cv-RA on the PLA2 activity induced by B. jararacussu crude venom (A),

Asp49-PLA2 BthTX-II (B) and Asp49-PLA2 BthA-I-PLA2 (C) at rations 1:5, 1:10 and 1:50 (venom:inhibitor, w/w). Results are expressed by

the meanGSD (nZ

6). Means are statistically significantly different (*) from the control means.




using Student’s unpaired t -test. A P-value !0.05 was

considered to indicate significance.

3. Results and discussion

In many countries, plant extracts have been traditionallyused in the treatment of snakebite envenomations (Martz,

1992; Mors et al., 2000; Soares et al., 2004a), although only

in a few cases there has been a scientific validation of such

claims. Snake venoms are complex mixtures of proteins and,

among these, are phospholipases A2, hemorrhagins,

proteases and myotoxins that act by different mechanisms

(Gutierrez, 2002). A number of PLA2s has been character-

ized from Bothrops venoms, some which are devoid of

catalytic activity upon artificial substrates due to the

substitution of Lys at position 49 for Asp (Soares et al.,

2004b).

Fig. 1 shows the purification of Cv-RA from theC. verbenacea methanolic extract. Cv-ME represented

2.25% (8.5 g) of the dried leaves. From its fractionation

on the Sephadex LH-20 column, three fractions were

obtained from which fraction 3 was less heterogeneous

and corresponded to 0.19% (0.7 g) of the dried leaves.

Among the seven subfractions resulting from the HPLC

rechromatography of fraction 3, subfraction 6 (CL-6)

showed to be highly purified (HPLC-C18) and represented0.03% (0.112 g) of the dried leaves. Spectroscopic analysis

of subfraction 6 identified it as rosmarinic acid. Its chemical

and tridimensional structure is shown in Fig. 2A and B,

respectively

Rosmarinic acid was first isolated from Rosmarinus

officinalis, but recently its synthetic preparation was

described. RA is often described as anti-inflammatory. It

is a polyphenolic compound, isolated from several plants of

Boraginaceae and Laminaceae families (Petersen and

Simmonds, 2003). This is the first report of rosmarinic

acid in the species C. verbenacea and explains the efficiency

of this plant regarding anti-inflammatory and antimyotoxicproperties against snake venoms and isolated toxins.

Fig. 5. Inhibition of the myotoxic activity by rosmarinic acid. Effect of Cv-RA on the myotoxic activity induced by B. jararacussu crude venom

(A), Lys49-PLA2 BthTX-I (B) and Asp49-PLA2 BthTX-II (C) at rations 1:1 and 1:10 (venom:inhibitor, w/w). Results are expressed by the

meanG

SD (nZ





Edema-inducing activity is a multifactorial pharmaco-

logical activity, depending on the combined action of

various toxins, suggesting that enzymatic activity is not

strictly required to induce this effect. Cv-ME inhibited near

20, 60 and 10% the edema induced by B. jararacussu crude

venom, BthTX-I and BthTX-II, respectively (Fig. 3A),while Cv-RA inhibited 5, 60 and 10% the edema induced by

these same samples (Fig. 3B). Cv-ME was more efficient in

neutralizing the edema induced by the crude venom than

Cv-RA, thus suggesting that other active principles are

presented in Cv-ME other than Cv-RA. An active flavonoid

component, artemetin, has previously been isolated from

this plant and shown to have anti-inflammatory effects

(Sertie et al., 1990).RA showed to be more efficient in neutralizing the PLA2

activity induced by the basic Asp49 BthTX-II (Fig. 4B) than

that induced by the crude venom (Fig. 4A) and by the acidic

isoform Asp49 BthA-I-PLA2 (Fig. 4C). These data suggest a

more specific binding with basic PLA2s, intermediated by a

probable electrostatic interaction. Biondo et al. (2003) also

showed that the aqueous extract from Mandevilla velutina

showed a wide inhibition spectrum of toxic, enzymatic and

pharmacological activities of snake venoms and isolated

toxins. However, this extract was more specific for Crotalus

venom and the neurotoxic basic PLA2 when compared with

Bothrops acidic PLA2.A partial dissociation between the catalytic and edema-

inducing domains is also likely to exist in these PLA2s, since

a 60% inhibition of the edema induced by the basic Lys49

BthTX-I, enzymatically inactive, was observed, against

only 10 and 50% inhibition of the edema and PLA2 activity,

respectively, induced by the basic Asp49 BthTX-II. These

data agree with several authors who suggest distinct

domains or partial overlapping between the catalytic and

other pharmacological sites (Soares and Giglio, 2003).

Muscle tissue damage, myonecrosis, is a common

consequence of envenomation by crotaline snakes of the

genus Bothrops (Gutierrez, 2002). Muscle damagingactivity of Bothrops venoms is partially caused by a group

Fig. 6. Analysis of B. jararacussu venom and isolated PLA2s,

BthTX-I and BthTX-II by SDS-PAGE 12%. Before incubation

with Cv-ME or Cv-RA: Lanes: 1, BthTX-II; 2, BthTX-I; 3,

B. jararacussu venom; 4, Cv-ME or Cv-RA. After incubation with

the Cv-ME or Cv-RA: Lanes: 1, Cv-RACBthTX-II; 2, Cv-RAC

BthTX-I; and 3, Cv-RAC B. jararacussu.

Fig. 7. Enhancement of the antimyotoxic properties of polyvalent anti-bothropic immunoglobulin antivenom by supplementation with Cordia

verbenacea rosmarinic acid (Cv-RA). Cv-RA was added to commercial polyvalent (Crotalinae) antivenom (Instituto Butantan, Brazil) at an

ED50 incubated with B. jararacussu venom or isolated PLA2s for 30 min at 37 8C, and injected intramuscularly in mice. Results are expressed

by the meanGSD (nZ





of highly basic proteins with PLA2 structure. Rosmarinic

acid inhibits the myotoxic activity of both Asp49 BthTX-II

and Lys49 BthTX-I phospholipases A2 from B. jararacussu

(Fig. 5). Rosmarinic acid did not inhibit the myotoxic

activity of the crude venom as effectively as that of the

purified PLA2s. This could very well be a result of the

contribution to myonecrosis made by the strong hemor-

rhagic toxins in the crude venom which are lacking in the

purified PLA2 preparations. A series of PLA2s inhibitors

(PLIs) has been isolated from natural sources, such as

marine organisms, snakes and plants (Lizano et al., 2003).

Wedelolactone and 12-methoxy-4-methylvoachalotine

(MMV), compounds isolated from Eclipta prostata and

Tabernamontana catharinensis, respectively, effectively

inhibits the myotoxic activity of the venoms of Crotalus

durissus terrificus, B. jararacussu, B. jararaca and Lachesis

muta, as well as various isolated myotoxic PLA2s (Mors

et al., 2000; Soares et al., 2004a).

Although the mechanism of action of C. verbenacea

methanolic extract (Cv-ME) and/or rosmarinic acid

(Cv-RA) is still unknown, the finding that no visible change

was detected in the electrophoretic pattern of B. jararacussu

venom, BthTX-I and BthTX-II, after incubation with Cv/RA

(Fig. 6), excludes proteolytic degradation as a potential

mechanism.

Preliminary studies on supplementation of conventional

antivenom against B. jararacussu or isolated myotoxic

PLA2s with the Cv-RA from C. verbenacea show that the

inhibitor enhances the neutralization potential of the

antivenom in mice (Fig. 7), which is often only partially

effective in neutralizing myotoxicity in vivo. As far as

toxicity is concerned, at least mice inoculated with

B. jararacussu venom and subsequently treated by

intravenous injection of the inhibitor or antivenom

immunoglobulins supplemented with Cv-RA show no

detectable signs of toxicity or adverse effects to the addition

of inhibitor. Similarly, adjuvant effects and antiserum action

Fig. 8. Analysis of circular dichroism spectra for RA in association

with BthTX-I. Spectra for the BthTX-I (open squares) alone or

BthTX-I and RA (closed squares), a mixture at a 1:3 molar ratio are

shown. The spectra shown are unsmoothed and corrected only by

subtraction of buffer blanks as described in Section 2.

Fig. 9. Molecular model of rosmarinic acid and monomeric BthTX-I complex. Drawn with the program RIBBONS (Carson, 1997). The residues

interacting with the rosmarinic acid and the BthTX-I are shown in ball-stick representation.




potentiation by a compound Hemidesmus indicus 2-hidroxy-

4-methoxy-benzoic acid against Vipera russelli venom were

described (Alam and Gomes, 1998).

Possible secondary structural changes either in the

Cv-RA or the target PLA2 following binding of the inhibitor

were evaluated by circular dichroism (CD) spectroscopy

(Fig. 8). The result for mixtures of the Cv-RA withLys49-BthTX-I was described, which reveal that the CD

spectra for the mixture of the two components is equal the

sum of the two individual spectra of the Cv-RA and the

BthTX-I alone. The experiments in which Asp49-BthTX-II

substituted for the BthTX-I yielded similar results (data not

shown), which suggests that no significant secondary

structure changes occurred on association of the Cv-RA

with the PLA2s tested.

In order to study the possible mechanism of Lys49-PLA2

BthTX-I inhibition by rosmarinic acid, a molecular model of

the complex was made. The rosmarinic acid was modeled

into the hydrophobic channel leading to the active site. Afterenergy minimization, the rosmarinic acid remained in the

hydrophobic channel with a hydroxyl group of one of the

aromatic rings bound to His48 and the carboxyl group

bound to the Lys69 (Fig. 9). This is a possible way for the

rosmarinic acid to interact with a phospholipase A2 leading

to its inhibition. His48 belongs to the catalytic network for

class II PLA2s being a strictly conserved residue to this class

of proteins. The majority of PLA2-inhibitor complexes have

these molecules bound to His48 (Watanabe et al., 2005),

which is seen to be essential for the inhibition process.

Lys69 is conserved residue in the most part of class II PLA2s

and is sited in a loop between a-helix 2 and b-wing known

as ‘pancreatic loop’. While, for Asp49-PLA2s, this residue is

associated with anticoagulant activities (Carredano et al.,

1998), no activity is associated with this residue for

Lys49-PLA2 until now.

The presence of PLA2 inhibitory proteins and other

compounds in plants opens the possibility to search for

natural inhibitors of snake venom myotoxic effects in plants

for therapeutic purposes. It is likely that other plants may

also serve as sources for PLIs that could be used in the future

as potent antivenom compounds.

4. Conclusions

Cv-ME and Cv-RA inhibit the edema and myotoxicity

induced by B. jararacussu crude venom and its main

phospholipases A2 homologs, thus showing that this plant is

a good tool with potential antiophidian activity. Cv-RA was

much more efficient to inhibit the edema induced by the Lys49

PLA2 BthTX-I than its isoform Asp49 BthTX-II. However, it

neutralized equally the myotoxicity induced by both toxins.

This fact suggests the presence of distinct domains for these

activities. Co-crystallization studies of this inhibitor with

Lys49 PLA2s are in progress for a better insight into the

mechanism of action of these enzyme and/or inhibitor.

Supplementation of antiophydian serum with natural

anti-toxins such as anti-hemorrhagins and anti-PLA2s could

increase the ability of serum to neutralize snake toxins. It is

interesting to speculate that Cv-RA, or a derivative, may

prove useful in the treatment of snakebite victims, or more

importantly in the treatment of the many human diseases in

which PLA2 enzymes have been implicated. In particular,the use of cell impermeable PLA2 inhibitors could be a

favorable therapeutic approach in the treatment of inflam-

matory processes.

Acknowledgements

The authors gratefully acknowledge the financial support

by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) and Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (CNPq). Thanks are also due to

Joa˜

o J. Franco (FCFRP-USP), Adelia C.O. Cintra (FCFRP-USP), Eliandra G. Silva (TT-FAPESP) and Vanessa

C. Fernandes (TT-FAPESP) for their helpful technical

collaboration.

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