Protein & Peptide Letters, 2012, 19, 501-508 501

Effects of Compounds from Passiflora edulis Sims f. flavicarpa Juice on Blood Coagulation and on Proteolytic Enzymes

Ana Claudia Satoa, Sonia A. Andrade

c, Marlon V. Brito

a, Antonio Miranda

b, Misako Uemura

Sampaioa, Francisco Humberto de Abreu Maffei

d,e and Maria Luiza Vilela Oliva

a,*

Departamento de aBioquímica,

bBiofísica, Escola Paulista de Medicina- Universidade Federal de São Paulo, São Paulo,

Brazil; cLaboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, Brazil;

dDepartamento de Cirurgia e

Ortopedia, Escola de Medicina de Botucatu - UNESP, Universidade Estadual de São Paulo, Botucatu, Brazil; eHospital

Santa Catarina, São Paulo, Brazil

Abstract: Passion fruit (Passiflora edulis Sims f. flavicarpa) is popularly known for its sedative and calming properties and is consumed as a fresh fruit or as a juice. The clinical observation of blood incoagulability associ-ated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted the current study to investigate in vitro the presence of blood clotting inhibitors in Passiflora edulis Sims f. flavicarpa extract.

After purification process, two compounds of distinct molecular weight and inhibitory action were better characterized.

One is a trypsin inhibitor similar to inhibitors from Bowman-Birk family, named PeTI-I12, and other is a compound ac-

tive in coagulation that prolongs aPTT and PT, but does not change TT. The aim of this study is to provide evidence that passion fruit extract’s components play a role on hemostasis and therefore may be relevant in the han-dling of patients treated with anticoagulants or suffering hemorrhagic diseases.

Keywords: aPTT, blood coagulation, Bowman-Birk inhibitor, Passiflora edulis, passion fruit, Passifloraceae, trypsin inhibitor, warfarin.

INTRODUCTION

The genus Passiflora consists of about 500 species [1-3]. Passiflora edulis Sims f. flavicarpa (P. eludis) which be-longs to the Passifloraceae family [4-6], is characterized by yellow fruits, acid pulp with rich flavor and smell, and is used for juice processing [6]. Leaves extracts were reported to be sedative and anxiolytic in mice [7, 8] and more recently their hypoglycemic and hypolipidemic properties were de-scribed [5, 9]. The bark has an antihypertensive effect in spontaneously hypertensive rats (SHR) due to the presence of -aminobutyric acid (GABA) and polyphenols with vaso-dilator properties, mainly luteolin [10]. Peel flour reduces total cholesterol, high-density lipoprotein cholesterol (HDL-C) and glucose levels [5, 9, 11], in humans. From the juice, studies reported anxiolytic-like activity, and flavonoids as one of the main active components [12]. Furthermore, the seeds were shown to have an antifungal peptide similar to 2S albumin family [13, 14, 15].

Recently, Carvalho et al. [16] verified that ethanol extract from leaves of Passiflora nitida Kunth, has anticoagulant activity as observed by prolongation of aPTT and inhibition of platelet aggregation. So far, it has not been identified a Passiflora’s component responsible for the pharmacological effects upon the coagulation cascade.

*Address correspondence to this author at the Universidade Federal de São

Paulo-Escola Paulista de Medicina, Departamento de Bioquímica, Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brazil;

Tel:/Fax: 551155736407; E-mail: olivaml.bioq@epm.br

The clinical observation of blood incoagulability associ-ated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted studies on the effect of the fruit extract on blood coagulation parameters and on enzyme inhibition. The present study describes the isolation and characterization of a trypsin inhibitor and peptide anti-coagulant from P. edulis Sims f. flavicarpa pulp juice.

MATERIAL AND METHODS

Bovine trypsin (EC 3.4.21.4) was from Calbiochem (San Diego, CA, USA) and factor Xa (EC 3.4.21.6) was from Sigma Chemical Company (St. Louis, MO, USA). Human plasma kallikrein (EC 3.4.21.34) had been purified as previ-ously described [17].

The substrates p-Nitroanilide (pNan) (Bz-Arg-pNan, Ac-Phe-Arg-pNan) and fluorogenic Boc-Ile-Glu-Arg-AMC were from Bachem (Torrance, CA, USA).

SP-Sephadex®

G-25 and CNBr-activated Sepharose were from GE Healthcare (Pittsburgh, PA, USA), and Jupiter 300 C18 was from Phenomenex (Torrance, CA, USA).

Actin Activated Cephaloplastin Reagent (activated partial thromboplastin time - aPTT), Thromborel S Reagent (prothrombin time - PT) and BC Thrombin Reagent (throm-bin time - TT) were from Dade Behring Inc. (Atlanta, GA, USA).

Passion fruits (Passiflora edulis Sims f. flavicarpa) pro-vided by a single producer, were acquired from a supermar-ket.

1875-5305/12 $58.00+.00 © 2012 Bentham Science Publishers

502 Protein & Peptide Letters, 2012, Vol. 19, No. 5 Sato et al.

Crude Extract Activities Characterization

Passiflora edulis Sims f. flavicarpa pulp (225 mL) was mechanically separated from the seeds and centrifuged at 9, 000 x g for 20 minutes at 4°C. After centrifugation, the pro-teins contained in the supernatant were precipitated with acetone 1:5 (v/v), kept at 4°C for 30 minutes. The sediment, separated by centrifugation, was dried at room temperature, identified as Pe-AF (P. edulis acetone fractionation), and dissolved in 0.1 M Tris solution (pH 11.0) to set the pH at 7.0. Trypsin inhibitory activity and the effect on coagulation cascade were performed under controlled pH and conductiv-ity.

To determine the heat stability of the trypsin inhibitor and the anticoagulant components, Pe-AF was treated at 100°C for 0-60 min. The temperature was slowly brought down to room temperature (25°C), and the residual enzy-matic and anticoagulant activities were determined respec-tively, as described bellow. All experiments were carried out in duplicate, and the results reported represent the mean value of two independent tests.

The soluble compounds in the Pe-AF was fractionated using dialysis steps against 250 mL of water for 12 h at 4°C through a 1000 Da molecular weight cut off membrane. The permeate and the retentate were concentrated by lyophiliza-tion, and protein concentration of the preparations was de-termined according to Lowry et al. [18].

Purification Process

After preliminary characterization of the crude extract, Pe-AF was submitted to size exclusion chromatography on a Sephadex G-25 column (60 cm x 1.5 cm) previously equili-brated with 5 mM Tris-HCl buffer, pH 7.0 (0.5 mL/min flow rate). The fractions containing trypsin inhibitor and antico-agulant activity were identified.

The trypsin inhibitory activity was applied to a trypsin-Sepharose column (2 cm x 1.5 cm), pre-equilibrated with 0.1 M Tris-HCl buffer, pH 8.0. After extensive washing with 0.1 M Tris-HCl buffer, pH 8.0 containing 0.3 M NaCl the inhibi-tor was eluted with 0.1 M HCl, pH 2.0 and subsequently neutralized with 0.1 M Tris-HCl buffer, pH 8.0. The affinity chromatography fraction that inhibits bovine trypsin was applied onto a C18 column using an HPLC system from Shi-madzu SLC-6A equilibrated with 0.1% (v/v) CF3CO2H (TFA) in water. Separation was achieved using a CH3CN gradient (0–100%, 60 min) in 0.1% (v/v) TFA (0.7 mL/min. flow rate) at room temperature. The eluted protein was used for amino acid sequence determination.

The anticoagulant activity eluted from Sephadex G-25 column was lyophilized, solubilized in 5 mM Tris-HCL buffer, pH7.0 and applied onto a C18 column using a HPLC system in the same conditions described for trypsin inhibitor.

Mass Spectrometry Determination

The LC/ESI–MS data of the trypsin inhibitor and antico-agulant was obtained on a Micromass instrument, model ZMD (Waters Corporations, Milford, MA), coupled to a Waters Alliance model 2690 system using a Waters photodi-ode array model 996, C18 column, solvent A: 0.1% TFA/H2O

and B: 0.1% TFA in CH3CN/H2O 60%, linear gradient 5-95% B for 60 minutes and 10 mL/min flow rate. The mass of the anticoagulant compound was obtained on a MALDI TOF/TOF 5800 (applied Biosystem) mass spectrum after purification on HPLC.

Structure Determination and Sequence Comparison

N-terminal sequence of the trypsin inhibitor was per-formed by the automated Edman degradation [19] method on a Shimadzu model PPSQ-23 Automatic Sequencer. Se-quence identity was determined using MEROPS database version 9.4 [20].

Enzyme Assays

The inhibitory activity of the crude extract or purified fractions on serine proteases was measured using specific chromogenic or fluorogenic substrates in 96-well microtiter plates at 37°C. The enzymes human FXa, trypsin and human plasma kallikrein were pre-incubated with crude extract, purified fractions or solvent, for 10 min. The reactions were initiated by the addition of the substrate, and the color or fluorescence was monitored continuously at 405 nm using a SpectraCount, or at 380/460 nm using a Hitachi F-2000 spectrofluorimeter, for 30 min, and stopped with the addition of 50 μL of 30% acetic acid. Enzymatic activity was assayed in buffers used to dilute the enzymes: human FXa (21.7 nM), in 20 mM Tris-HCl buffer, pH 7.4, containing 140 mM NaCl, 5 mM CaCl2, and 0.1% serum albumin; trypsin (0.038 μM) in 0.1 mM Tris-HCl, pH 8.0 containing CaCl2 0.02% (v/v); human plasma kallikrein (HuPK) (12 μM) in 0.1 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl.

Kiapp Determination for Trypsin Inhibition

The equilibrium dissociation constant (Kiapp) was deter-mined for trypsin through pre-incubation of the enzyme with increasing concentrations of the inhibitor at 37°C in 50 mM Tris-HCl buffer, pH 8.0 containing CaCl2 0,02% (v/v), in a final volume of 250 μL. Residual activity was subsequently measured using 1 mM Bz-Arg-pNan as substrate. Apparent Kiapp was determined by fitting the experimental points to the equation for slow-tight binding [21] with the help of the Grafit program, version 4.0 (Erithacus Software, Staines, UK).

Temperature stability- The inhibitor was dissolubilized in 50 mM Tris-HCl buffer, pH 8.0 and incubated at various temperatures of 0, 37, 50, 60, 80, and 100 °C for 10 min. Assay of trypsin inhibitory activitywas carried out as de-scribed above.

Plasma Preparation and Coagulation Assays

Human blood from apparently healthy donors (10 volun-teers, aged 25–30 years not subjected to treatment with anti-coagulant or anti-platelet drugs), who were informed about the study and signed an informed consent before starting the research, was collected by venipuncture in a tube containing 3.2% sodium citrate (BD Vacutainer). The aPTT assay was carried out according to the manufacturer specifications (Dade Behring). Pooled, citrated, normal human plasma was briefly mixed with the crude extract or fractions after reverse

Passion Fruit Proteinase Inhibitor Protein & Peptide Letters, 2012, Vol. 19, No. 5 503

phase chromatography at different concentrations. The aPTT reagent (50 L) was added to the mixture and incubated for 2 min at 37°C. Thereafter, 0.025 M CaCl2 (50 L) was added and the clotting time was recorded. The PT assay was per-formed using the PT reagent (lyophilized thromboplastin from human placenta). Normal plasma was pre-incubated with the extract or fraction at different concentrations for 1 min at 37°C. Then, 100 L of PT reagent was added, and clotting time was measured. Thrombin time (TT) was meas-ured by pre-incubating the normal plasma with the extract or fraction at different concentrations for 2 min at 37°C. The reaction was activated by adding 200 L of bovine thrombin (3.0 UNIH/mL), and the clotting time was measured. The assays were made in duplicate and the results expressed by the average of the determinations of each sample using the semi-automatic BFTII Dade Behring coagulometer.

RESULTS AND DISCUSSION

Crude Extract Activity Characterization

Prior to the coagulation and enzyme activity assays, the pH of P. edulis extract was set at 7.0 since the low pH of the juice may alter the coagulation parameters, as well the en-zyme activity on synthetic substrates. Studies on pH depend-ence of serine proteases such as trypsin, chymotrypsin and thrombin demonstrate that these serine proteases exhibit two

ionizations important for expression of enzymatic activity: one at a neutral or slightly acidic pH (pH 6-7.5) and a second at a more basic pH (pH 8-9) [22, 23]. The pre-incubation of the extract with poor platelet plasma leads to a significant prolongation of activated partial thromboplastin time (aPTT) and prothrombin time (PT), data not shown, suggesting that P. edulis played a role in anticoagulation by inhibiting intrin-sic and extrinsic coagulation factors, however, it did not af-fect thrombin time (TT). Trypsin activity was slightly inhib-ited providing evidence for the trypsin inhibitor in the pas-sion fruit juice.

In order to characterize the compound(s) responsible for the anticoagulant activity, the extract was concentrated by acetone precipitation. Commonly used to precipitate pro-teins, acetone fractionation [24-26] was efficient for concen-trating the proteins from P. edulis juice (Pe-AF) and useful in detection of trypsin inhibition. Indeed, the use of cold ace-tone was important and promoted the elimination of a large proportion of lipids and other components that can interact with proteins. In addition, this solvent is easily removed from the crude extract preparation.

Pe-AF prolonged aPTT Fig. (1A) and PT Fig. (1B) in a dose-dependent manner, but did not inhibit TT (data not show), indicating that the thrombin activity is not altered by the components of the fruit extract. The Fig. (1C) shows the heat stability of Pe-AF anticoagulant activity. After boiling

Figure 1. (A) Pe-AF effects ( g – Lowry et al., 1951) on aPTT. The aPTT was determined at 37°C after the addition of cephalin CaCl2 on

citrated plasma with and without P. edulis Sims f. flavicarpa. UNF: unfractionated heparin. (B) Effect of Pe-AF on PT. The PT was deter-

mined at 37°C after the addition of thromboplastin on citrated plasma with and without P. edulis Sims f. flavicarpa. (C) Pe-AF was pre-

heated at 100°C for different periods of time and then kept at room temperature. After that, the aPTT was performed.

504 Protein & Peptide Letters, 2012, Vol. 19, No. 5 Sato et al.

for 10 min, the anticoagulant activity remains, demonstrating high stability, but there were a total losses of activity under heat treatment at 100°C for 20 min Fig. (1C). Heat-stable components of low molecular weight that interfere with he-mostatic system have been characterized from human diet [27]. To heat the crude extract is advantageous because this procedure leads to the denaturation of enzymes, thus preven-ting the proteolytic degradation of the compounds of interest.

The anticoagulant activity was not more detected after extensive dialysis of the Pe-AF with water, indicating that a component of low molecular weight in passion fruit juice interferes with blood coagulation, while the trypsin inhibi-tory activity was detect in the retentate fraction.

Inhibitor Purification

For purification of the inhibitory activities, the passion fruit juice was submitted to several procedures that com-bined heating (10 min, 60 °C), protein precipitation, size exclusion, affinity, and reverse-phase chromatographies.

Resins with different size exclusion ranges were tested and the best procedure used for the preparation of the inhibi-tor was Sephadex G-25, in which compounds with lower molecular mass were retained longer in the pores of resin allowing removal of any pigment still remaining. This pro-cedure facilitates enzyme inhibition studies with synthetic substrates, especially in colorimetric reactions. The profile of Pe-AF after size exclusion chromatography on Sephadex G-25 column is shown in Fig. (2A). The purified proteins poorly absorbed at 280 nm, probably due to low aromatic amino acid residue content. The fraction A showed trypsin inhibitory activity, but did not interfere with aPTT and PT coagulation assays. Under these conditions, this fraction was purified overall 8-fold and the yield was 36%. The molecular mass of fraction B further purified by C18- reverse phase chromatography ranged from 870.97 to 923.16 Da Fig. (2B). It did not demonstrate trypsin and HuPK inhibitory activi-ties, however, it increased aPTT and PT, but did not change TT. Prolongation of aPTT in FVIII-deficient plasma and no HuPK inhibition indicates that the mechanism by which the active principle acts on coagulation does not involve the in-hibition of these factors.

In order to confirm the presence of a distinct form of trypsin inhibitor, the fraction A was further chromatographed on trypsin-Sepharose and activity was recovered in a peak identified as fraction Z Fig. (2C). The homogeneity of the inhibitor was assessed by reverse phase chromatography in C18 column with an acetonitrile gradient from which the pro-tein eluted at around 10.90 min Fig. (2D). The same material was used for sequence determination.

Trypsin Inhibitor Structural and Functional Characteri-zation

The PeTI-12 molecular mass 13902 Da is a number-average from 13380 to 14425 Fig. (3A), and the search of N-terminal region sequence using MEROPS database Fig. (3B) showed that the inhibitor is closely related to Bowman-Birk group I-12 [20] inhibitors, such as those from Dioclea glabra

[28], Canavalia lineata [29], Dipteryx alata [30], Lens culi-naris [31] and Vigna mungo [32], justifying its denomination PeTI-I12.

BBIs are divided into several protein families according to their amino acid sequence similarity, the nature of the inhibitory domain(s) and the mechanism of their interaction with proteases. Studies on the evolutionary aspects of this group of proteins found in various species of the monocoty-ledonous grass family (Poaceae), dicotyledonous legume species (Leguminosae family), including soybean (Glycine max), chickpea (Cicer arietinum), common bean (Phaseolus vulgaris), lentil (Lens culinaris) and pea (Pisum sativum), and a small Bowman-Birk like inhibitor isolated from sun-flower (Helianthus annuus) seeds suggest that BBIs have probably evolved from an ancestral single headed inhibitor by internal gene duplication and following mutations within the active site domains. Thus, Bowman-Birk type is rather variable and can be distinguished by two groups: the lower molecular weight group (about 8 kDa), with a single active site, and the higher molecular weight (about 16000 Da) group, possessing two active sites and also a high cysteine content [33]. PeTI-I12 molecular weight is similar to Bow-man-Birk type II inhibitors. The short N-terminal region found in PeTI-I12 needs to be understood, it may be result from proteolytic cleavage since more than one peak was seen by mass spectrometry determination. As it was focalized by the studies of many groups, post-translational modifications at the N- and C-terminal ends occur. Taking into account these facts, it can be assumed that some residues from N-terminal segment may have been lost by the PeTI-I12 inhibi-tor during evolution.

The affinity of the PeTI-I12 interaction with trypsin was investigated by kinetic measurements at various concentra-tions of inhibitor. In this case, it was necessary to fit the ac-tive enzyme concentration, because the procedure requires an exact knowledge of the active-site concentration. The exactly concentration of trypsin active site concentration was determined by NPGB titration [34]. PeTI-I12 is very effec-tive for bovine trypsin inhibition. The low Kiapp value (2.3 nM), calculated using the equation described by Morrison et al. [35], and indicates high-affinity binding inhibitory mechanism described for BBIs [33]. No delay of blood co-agulation time parameters or inhibition of synthetic substrate hydrolysis by the enzymes HuPK and factor Xa were de-tected. Inhibitors from vegetal origin are able to block co-agulation enzymes, such as plasma kallikrein [25], and factor Xa inhibitors [24], but none of them belongs to the Bowman-Birk family. No inhibition of plasma, tissue kallikrein, nor thrombin has been reported for these inhibitors. The stoichiometry of PeTI-I12 and trypsin complex in solution was investigated, and the inhibition was approximately linear with increasing concentration PeTI-I12, and at an approxi-mate molar ratio of inhibitor: trypsin (1:1), trypsin activity was completely inhibited Fig. (4A) i.e., one molecule of in-hibitor binds one molecule of enzyme, suggesting that PeTI-I12 has a single inhibitory site for trypsin inhibition. Thus, PeTI-I12 molecular weight and specificity is similar to the specificity of Bowman-Birk type II inhibitors, all specific trypsin inhibitors.

Passion Fruit Proteinase Inhibitor Protein & Peptide Letters, 2012, Vol. 19, No. 5 505

Figure 2. (A) Size exclusion chromatography on Sephadex G-25 column. Sample: 500 g of Pe-AF (Lowry et al., 1951). The chromatogra-

phy was performed with 5 mM Tris-HCl buffer, pH 7,0 and flow rate 0.5 mL/min. Fraction A: trypsin inhibitor measured by trypsin (37 nM)

hydrolysis of the substrate Bz-Arg-pNan (1.0 mM) in 0.1 M Tris-HCl buffer, pH 8.0, containing 0.02 % CaCl2, Fraction B: aPTT and PT

prolongation activity. The aPTT was determined at 37°C after the addition of cephalin CaCl2 on citrated plasma with the fraction B. The PT

was determined at 37°C after the addition of thromboplastin on citrated plasma with the fraction B. Fraction C: no activity on trypsin, aPTT

or PT. (B) MALDI TOF/TOF 5800 (applied Biosystem) mass spectrum from inhibitor of the blood coagulation after purification on HPLC.

(C) Trypsin-Sepharose chromatography. The fraction A from size exclusion chromatography was submitted to trypsin-Sepharose column

equilibrated with 0.1 M Tris-HCl buffer, pH 8.0 following the elution with 0.1 M Tris-HCl, pH 8.0 containing 0.3 M NaCl and afterwards, by

acidification with 0.1 M HCl pH 2.0. The samples eluted by the acid were immediately neutralized with 1 M Tris-HCl buffer, pH 8.0 and the

trypsin inhibitory activity determined as described. (D) Reverse phase HPLC C18 column was previously equilibrated with TFA 0.1% (v/v)

and eluted with gradient increasing ACN 60% (v/v) in TFA 0.1% (v/v), 0.7 mL/min. flow rate at room temperature.

506 Protein & Peptide Letters, 2012, Vol. 19, No. 5 Sato et al.

Figure 3. (A) The LC/ESI–MS data of the trypsin inhibitor was obtained on a Micromass instrument, model ZMD (Waters Corporations,

Milford, MA), coupled to a Waters Alliance model 2690 system using a Waters photodiode array model 996. (B) Sequence similarity of P.

edulis inhibitor with other inhibitors from plant. (1) P. edulis trypsin inhibitor-PeTI; (2) Diocleia glabra trypsin inhibitor-DgTI (Bueno et al.,

1999); (3) Canavalia lineata trypsin inhibitor-ClTI (Terada et al., 1994); (4) Dipteryx alata trypsin inhibitor-DATI (Kalume et al., 1995); (5)

Lens culinaris trypsin inhibitor-LCTI (Ragg et al., 2007); (6) Vigna mungo-VM (Prasad et al., 2010). The dashes indicate gaps which were

introduced for optimal alignment and maximum similarity for the MEROPS database. Residues identical to PeTI-I12 are displayed in black

boxes. Trypsin Inhibitor Stability Characterization

The study of the temperature effect (0-100°C) showed that the inhibitory activity of PeTI-12 was maintained at tem-peratures up to 60°C for 10 min but not at 100 °C Fig. (4B). Since variation in temperature affects the strength of weak

weak bonds, and therefore can alter multiple protein proper-ties including conformation and affinity for ligand substrates [36], the thermolability demonstrated by these activities sug-gested a protein nature of the studied compounds. Structur-ally, Bowman-Birk inhibitors lack -helix [37], its disulfide

Passion Fruit Proteinase Inhibitor Protein & Peptide Letters, 2012, Vol. 19, No. 5 507

linkages minimize their conformational entropy and enhance their stability [38]. An example of this stability is described for the cyclic peptide isolated from sunflower, SFTI-1, that displays activity against trypsin and several other serine pro-teases, including thrombin, a protease target for anti-thrombotic drug development [39].

Figure 4. Trypsin inhibition by PeTI-I12. (A) Bovine trypsin (42

nM) preincubated (10 min, at 37°C, 0.1 M Tris-HCl buffer, pH 8.0)

with increasing amounts of PeTI-I12 eluted from trypsin-Sepharose

chromatography. The residual trypsin activity was assayed on Bz-

Arg-pNan (1.0 mM) as substrate, as described in Methods. (B) Ef-

fect of heat treatment at different temperatures on trypsin inhibitory

activity PeTI-I12. Bar indicate SD from duplicate determination.

Many studies attributed to BBIs antibiotic and anti-parasitic actions to their interference with protein digestion, decreasing the amount of available amino acids and thus delaying the synthesis of proteins necessary for reproduction, development and growth, and invasion of the pathogens [40, 41, 42]. In the case of PeTI-I12 it heat-lability may be impor-tant for homeostasis of protein metabolism, i.e. the intake of passion fruit juice will not interfere in the assimilation of nutrients impairing digestion and absorption of essential amino acids.

Considering the small size, high inhibitory efficacy and structural stability of BBIs, its biotechnological potential has been intensely investigated, and several research groups have suggested that these proteins may serve as a scaffold for the design of novel peptide-based drugs [33]. The potential for serine protease activity during tumour development has stimulated various lines of research. For example, Chu et al. (1997) [43] investigated the proteolytic activities of transfor-med cells inhibited by BBIs. In addition, Garcia-Gasca et al. (2002) [44] analysed the action of Phaseolus acutifolius BBI on in vitro cell proliferation and cell adhesion of transformed cells, and Kennedy and Wan (2002) [45] e mais recentes) evaluated the anti-carcinogenic activity of soybean BBIs on prostate cancer cells.Thus, characterization of this type of protein in P. edulis juice may stimulate its intake in human nutrition as well as their use as tools for pharmacological studies.

In conclusion, we found on the extract of Passiflora edulis Sims f. flavicarpa two compounds with distinct in-hibitory action: a thermostable anticoagulant, which structure is under investigation, and a very stable and highly potent B. Birk-like trypsin inhibitor. The current information may be relevant in the handling of patients with coagulation dys-function or hemorrhagic diseases.

ACKNOWLEDGEMENTS

This work was supported by Fundação de Amparo à Pes-quisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal e Nível Supe-rior (CAPES). We thank the technical assistance of Luci-meire A. de Santana and Izaura Yoshico Hirata and the Eng-lish revision of Scott V. Heald.

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Received: June 21, 2011 Revised: January 24, 2012 Accepted: January 24, 2012