Browplasminin, a condensed tannin with anti-plasmin activity isolated from an aqueous extract of Brownea grandiceps Jacq. flowers

Journal of Ethnopharmacology

Available online 12 January 2017

In Press, Accepted Manuscript — Note to users

• Betzabeth Pereira^a,
• Josmary Brazón^{a, , ,} ,
• Mónica Rincón^b,
• Eva Vonasek^b

• ^a Laboratorio de Neurofarmacología Celular, Centro de Biofísica y Bioquímica
• ^b Unidad de Proteómica, Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC). Apartado 20632 Caracas, 1020-A. Venezuela

Received 3 October 2016, Revised 26 December 2016, Accepted 9 January 2017, Available online 12 January 2017

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http://dx.doi.org/10.1016/j.jep.2017.01.012

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Highlights

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Species from Brownea genus are used in the traditional medicine for treatment of hemorrhages.

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Females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers decoctions to reduce the bleeding.

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This is the first work about a condensed tannin (called Browplasmin) that present anti-plasmin effect with an activity as effective as reference compounds used for treatment of menorrhagia like tranexamic acid.

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Abstract

Ethnopharmacological relevance

Following Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity.

Materials and methods

A decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics.

Results

The active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000 Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC₅₀ for plasmin was 47.80 μg/mL and for FXa was 237.08 μg/mL, while the Ki were 0.76 and 61.61 μg/mL for plasmin and FXa, respectively.

Conclusions

The overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.

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Graphical abstract

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Abbreviations

• BGE, Brownea grandiceps Jacq. flowers aqueous extract;
• HPLC, high-performance liquid chromatography;
• MALDI-ToF, matrix-assisted laser desorption/ionization - time of flight;
• MS, mass spectrometry;
• FXa, factor X activated;
• t-PA, tissue plasminogen activator;
• u-PA, urokinase plasminogen activator;
• TA, tranexamic acid;
• EACA, ε- aminocaproic acid;
• g, grams;
• g, gravity;
• TFA, trifluoroacetic acid;
• ACN, acetonitrile;
• DHB, 2,5-Dihydroxybenzoic acid;
• CI, confidence interval

Chemical compounds studied in this article

• Aprotinin (PubChem CID: 22833874);
• Benzamidine (PubChem CID: 80289);
• ε-aminocaproic acid (PubChem CID: 564);
• Tranexamic acid (PubChem CID: 5526);
• Acetonitrile (PubChem CID: 6342);
• Trifluoroacetic acid (PubChem CID: 6422);
• Ammonium acetate (PubChem CID: 517165);
• Sodium chloride (PubChem CID: 5234);
• Cesium chloride (PubChem CID: 24293);
• Calcium chloride (PubChem CID: 5284359);
• Trizma base (PubChem CID: 6503);
• Hydrochloride acid (PubChem CID: 313);
• Imidazole (PubChem CID: 795)

Keywords

• Brownea;
• coagulation;
• tannin;
• fibrinolysis;
• galloylation;
• proanthocyanidins

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1. Introduction

The menstrual bleeding of women in reproductive age is produced by an activation of local fibrinolysis in the endometrium, which depends on the balance between production and release of plasminogen activators and inhibitors. Endometrial fibrinolysis is excessive in women with heavy menstrual blood loss (menorrhagia), because there are elevated levels of endometrium derived plasmin and plasminogen activators that result in an increased local fibrinolysis (Philipp, 2011). In surgeries with high risk of blood loss such as cardiac, liver, tooth extraction as well as in menorrhagia treatments, plasmin (trypsin-like serine protease that degrades fibrin) is an important target to reduce bleeding (Lien and Milbrand, 2006, Schouten et al., 2009 and Philipp, 2011).

Multiple pharmacological approaches have been proposed in an attempt to attenuate the undue fibrinolytic system activation and to decrease blood loss. Some synthetic or natural molecules such as trans-4-aminomethylcyclohexane-carboxylic acid or tranexamic acid (TA), 6-aminohexanoic acid or ε- aminocaproic acid (EACA) and aprotinin are used as therapeutic agents in plasmin inhibition (Mannucci, 1998, Munoz et al., 1999 and Tengborn, 2012). TA (157 Da) and EACA (131 Da) are plasmin synthetic inhibitors, while aprotinin (6.512 Da) is a natural inhibitor. The first two, are lysine analogous that act as reversible inhibitors by competitively blocking the lysine-binding site of plasminogen/plasmin, thus preventing its attachment to fibrin (Tengborn, 2012), while aprotinin is a polypeptide of 58 residues, isolated from bovine lung, that directly inhibits plasmin and other serine-proteases (Mannucci,1998). Aprotinin is not used to reduce heavy menstrual blood loss anymore; as TA is more potent than EACA to treat menorrhagia, it is the main therapeutic alternative to control this disorder now-a-days (Mannucci, 1998 and Tengborn, 2012).

TA can reduce menstrual bleeding by approximately 50% and in most studies it is administered from days 1 to 4 or 5 of menses, in a dose of 4 g/day, during 3 consecutive menstrual cycles. However, nausea, vomiting, diarrhea, and some impaired color vision has been reported after the administration of TA (Philipp, 2011).

In most developing countries, plant remedies are the most prevalent treatments, where recipes have been handed down from generation to generation and every culture has used decoctions or extracts of leaves, flowers, barks or roots to treat various medical problems as hemorrhages, wound healing, menstrual disorder between others (Agra et al., 2007, Díaz and Ortega, 2006, Klitgaard, 1991a, Klitgaard, 1991b and Otero et al., 2000).

Brownea genus, comprising evergreen small trees with capitate racemes axillary or terminal of red, yellow or white flowers; which are found from the south of Mexico to Peru, including Antillean islands, Jamaica, Trinidad and Tobago ( Agra et al., 2007, Bussmann and Sharon, 2006 and Klitgaard, 1991a). Species from this genus are used in the traditional medicine for treatment of hemorrhages ( Díaz and Ortega, 2006, Márquez et al., 2005 and Otero et al., 2000). In Colombia and Brazil, some plant species from this genus such as B. ariza Bentham and B. rosademonte, are mostly employed in folk medicine as cicatrizant and to neutralize hemorrhages caused by snake venom ( Otero et al., 2000). However, in Venezuela and Ecuador, flowers of B. grandiceps Jacq. (popularly known as mountain rose or rose of Venezuela) are consumed as decoction in traditional medicine to reduce the bleeding in females with heavy menstrual blood loss ( Díaz and Ortega, 2006, Klitgaard, 1991a, Klitgaard, 1991b and Pereira and Brazón, 2015).

Women with menorrhagia inform that decoction of Brownea grandiceps Jacq. flowers (BG) increases their quality of life because this clinical problem alters their emotional, social and working life (personal communication). In a previous study, we demonstrated that BG aqueous extract possesses a potent anti-fibrinolytic activity that inhibits plasmin and which is also a rich source of bioactive compounds with activity on other human proteases ( Pereira and Brazón, 2015). As until now, an anti-plasmin component had not been isolated from BG, the aim of this work was to purify and characterize the plasmin inhibitor from Brownea grandiceps flowers aqueous extract.

2. Materials and methods

2.1. Reagents

Ammonium acetate (CH₃COONH₄), acetone, NaCl, CsCl, aprotinin, ε- aminocaproic acid (EACA), tranexamic acid (TA), benzamidine, imidazole, trypsin (ref. T1426-1G) and thrombin (ref. 605190-1000U) were obtained from Sigma-Aldrich^® (St. Louis, MO, USA). trifluoroacetic acid (TFA) and acetonitrile (ACN) for HPLC were purchased from Merck (Massachusetts, USA). DHB and calibration solution for mass spectrometry were acquired from Bruker Daltonik GmbH (Bremen, Germany). Fibrinogen, chromogenic substrates: S-2251 (H-D-norleucyl-cyclohexylalanyl-lysine para-nitroanilide diacetate salt, ref. 251 L) for plasmin, S-2222 (Methoxycarbonyl-D-cyclohexylglycylglycyl-arginine-para-nitroanilide acetate, ref. 222 L) for factor X activated (FXa) and trypsin, S-2288 (Methylsulfonyl-D-cyclohexyltyrosil-glycyl-arginine paranitroaniline acetate, ref. 444 L) for tissue plasminogen activator (t-PA) and S-2444 (carbobenzoxy-L- γ-glutamyl(α-t-butoxy)-glycyl-arginine paranitroaniline monoacetate salt, ref. 244 L) for urokinase plasminogen activator (u-PA), as the enzymes: plasmin (ref. 411), FXa (ref. 526), t-PA (ref. ADG170) and u-PA (ref. 128) were purchased from Sekisui Diagnostics (Lexington, USA).

2.2. Collection of plant material and preparation of aqueous extract

Brownea grandiceps Jacq. flowers were collected from 3-m-tall wild trees, fertilizers free, in the areas surrounding the Posada Mocundo, Carabobo State, Venezuela (10° 14’ 14’’ N–68° 15’ 44’’ W). The identity of this plant, which was collected in March 2014, was confirmed by Reina Gonto and a voucher specimen (number 5699) was deposited in the herbarium of the Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.

The Brownea grandiceps flowers aqueous extract (BGE) was prepared from a decoction using 229 g of Brownea grandiceps fresh flowers (1 inflorescence) in 2.5 L of double distilled water at 100 °C for 40 min which was then homogenized in a blender during 5 min. The homogenate was centrifuged at 27.500 g for 30 min at 4 °C; the supernatant was lyophilized, weighed (23 g) and stored at 4 °C until use.

2.3. Purification of plasmin inhibitor

An anti-plasmin inhibitor was isolated from a solution of 6 mg BGE in 100 µL of 20 mM CH₃COONH₄ pH 4.7, using a Protein Pack 300^TM column (7.8×30 cm, Waters, USA) coupled to a Waters HPLC 2695 with a quaternary pump and a photodiode array detector model 2996. Elution of BGE components was performed at a 0.5 mL/min flow rate under isocratic conditions with 20 mM CH₃COONH₄ pH 4.7 during 60 min at room temperature (20 °C) and detected at a wavelength of 280 nm. Fractions were manually collected and used to determine the inhibitory activity of plasmin. The active fraction (3.21 mg in 100 µL of HPLC water) was applied to an analytic C18 reverse phase column (4.6×250 mm, Vydac, USA) equilibrated with 0.12% TFA in water and eluted with a linear gradient of solvent A (0.12% TFA in water) to 60% of solvent B (0.1% TFA in ACN). The flow rate was 1 mL/min, and the process was monitored at a wavelength of 230 nm during 60 min. The anti-plasmin fraction (0.63 mg in 100 µL water) was re-purified through the same column with a linear gradient of 18 to 27% solvent B in 20 min. The active fraction was then called Browplasminin and was freezed-dried and stored at −20 °C until use.

2.4. MALDI-ToF analysis

The spectra were recorded on a Bruker MALDI-ToF/ToF mass spectrometer Autoflex III Smartbeam (Bruker Daltonics, Germany) provided with a smart beam laser (200 Hz). The mass spectra were acquired in both positive and negative ion mode, over a mass range of 400–5000 Da, with an acceleration voltage of 20 kV and a reflectron voltage of 23 kV; DHB was used as matrix. For analysis in negative mode, Browplasminin was resuspended in water (10 mg/mL). Browplasminin was mixed with a solution of DHB (20 mg/mL in 30% ACN, 0.01% TFA) at equal volumes. Then, 4 μL of the resulting mixture was placed on a MALDI steel target. Alternatively, to promote the formation of ion adducts [M+Na⁺] or [M+Cs⁺] in positive mode, Browplasminin was dissolved in acetone 30% (20 mg/mL) and it was mixed with DHB in the same solvent (20 mg/mL) at equal ratios. Two microliters of NaCl (1.5 mg/mL) or CsCl (1.520 mg/mL) were added to the sample/matrix solution and 4 μL of this mix were spotted.

2.5. Inhibition of plasmin amidolytic activity

The effect of BGE and its fractions on plasmin amidolytic activity was evaluated by the method of Guerrero and Arocha-Piñango (1992). The assay was carried out in 96-well polystyrene plates (Nunc^TM, USA), containing a mixture of 60 µL of recommended buffer for the chromogenic substrate (S-2251), 20 µL of plasmin (0.652 µM) pre-incubated at 37 ^oC for 30 min with BGE (100 µg/mL), fractions (100 µg/mL) or Browplasminin (6.25, 12.5, 25, 50 and 100 µg/mL) and then, 20 µL of S-2251 (0.8 mM) was added. After, the plate was incubated at 37 ^oC for 30 min and the absorbance at 405 nm was measured. The results were expressed as residual amidolytic activity percentage. The positive controls were aprotinin (100 µg/mL), EACA (100 µg/mL) and TA (100 µg/mL).

2.6. Inhibitory effect on other serine-proteases

To determine the specificity of the anti-plasmin component, the effect of Browplasminin was also evaluated on trypsin, FXa, t-PA and u-PA amidolytic activity, using a similar protocol as described above. The concentrations of FXa, t-PA, u-PA and trypsin, were 0.7 µM, 1.38 µM, 1 IU/µL and 0.08 µg/µL, respectively. The chromogenic substrates for the enzyme mentioned and the concentrations used were: S-2222 (0.8 mM), S-2288 (1.2 mM) and S-2444 (1 mM) respectively. The results were expressed as residual amidolytic activity percentage. Aprotinin (100 µg/mL), EACA (100 µg/mL), TA (100 µg/mL) and Benzamidine (10 mM) were used as controls.

2.7. Anti-fibrinolytic Activity

Anti-fibrinolytic activity was evaluated by the fibrin plate method (Marsh and Arocha-Piñango, 1972). The fibrin films on 3.5 cm plates were prepared, incubating at 25 °C for 30 min 1.5 mL of 0.1% fibrinogen (containing 10% of plasminogen as contaminant) in imidazole saline buffer and 75 µL of bovine thrombin (10 IU/mL, in 0.025 M CaCl₂). Then, ten microliters of a mixture of plasmin (0.652 µM) with BGE, fractions or Browplasminin (100 µg/mL), pre-incubated at 37 °C for 30 min, were applied over the films. Such plates were then incubated at 37 °C for 24 h. The diameters of the lysed areas were measured and the residual fibrinolytic activity was expressed in mm². Imidazole saline buffer, plasmin, aprotinin (100 µg/mL), EACA (100 µg/mL) and TA (100 µg/mL) were used as controls.

2.8. IC₅₀ Calculation

To determine the concentration of Browplasminin required to inhibit 50% of the amidolytic activity (IC₅₀) of plasmin or FXa, the IC₅₀ was calculated from fitting the dose-response data using a logarithmic non-linear regression analysis performed using the LibreOffice Calc version 4.0.3.3 (The Document Foundation, Germany).

2.9. Kinetics of plasmin inhibition

The IC₅₀ value was also determined directly by the simple method of Copeland et al., 1995, in which the dissociation constant (Ki) value of tight-binding inhibitors of enzymes can be calculated from graphical analysis of the dose-response curve. This alternative method, allowed the construction a dose-response plot as a function of different inhibitor concentrations (6.25, 12.5, 25, 50 and 100 µg/mL) using fixed concentrations of plasmin (0.652 µM) and S-2251 (0.8 mM) as substrate.

The data for the concentration dependent inhibition were analyzed with Eq. (1):

equation(1)

$\mathcal{V}/{\mathcal{V}}_o=1/1+(\left[I\right]/I{C}_{50})$

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Where V and V_ο are the velocities of the plasmin catalyzed reaction in the presence and absence of Browplasminin respectively, [Ι] is the concentration of Browplasminin present, and ΙC₅₀ is that concentration of Browplasminin required to reduced the reaction velocity to one half of that observed in its absence.

When [Ι] = ΙC₅₀, the value of V / V_ο is 0.5 and the value of V_ο / V must be 2.0, then the Ki value of competitive tight-binding inhibitors may be expressed in terms of the Eq. (2):

equation(2)

${\text{Ki}=(\text{IC}}_{50}-\left[\mathrm{E}\right]/2)/\left(\text{1}+\left[\mathrm{S}\right]/\text{Km}\right)$

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Where [S] and [E] are the concentrations of S-2251 and plasmin respectively and Km is the Michaels constant for the chromogenic substrate (S-2251).

2.10. Statistical analysis

Since the sample size (n=3) was small and the distribution of data unknown, the use of nonparametric statistical procedures was mandatory (Colquhoun, 1971). Data are presented as medians and their 90% confidence interval (CI) calculated with the procedure of Hodges and Lehmann (Hollander and Wolfe, 1973).

3. Results and discussion

3.1. Isolation of the plasmin inhibitor

The aqueous extract of Brownea grandiceps flowers (6 mg in 100 µL water) was fractionated by HPLC using the Protein Pak 300^TM semi-preparative size exclusion column under isocratic conditions, four fractions were collected and denominated F1 – F4 as depicted in Fig. 1A. The last fraction (F4) showed the highest activity of all on plasmin. Fraction F4 (3.21 mg in 100 µL water) was further chromatographed by analytical reverse phase HPLC using a C18 column (Fig. 1B) and it was separated in 8 new fractions with retention times between 12.88–30.00 min, these fractions were denoted F4-a – F4-h. Anti-plasmin activity was detected in the F4-h fractions which eluted at 21.10 min, this fraction (0.63 mg in 100 µL water) was rechromatographed in the same column and the active fraction that eluted at 5.78 min was named Browplasminin (Fig. 1C).

Fig. 1. 

Purification of Browplasminin from aqueous extract of Brownea grandiceps flowers (BGE) by HPLC.1.- Size exclusion chromatography of BGE (6 mg in 100 µL water) on a Protein Pack 300 column (7.8×30 cm) equilibrated and eluted with CH₃COONH₄, pH 4.7 at 0.5 mL/min. Four fractions were collected (F1-F4). 2.- Reverse-Phase chromatography of F4 fraction (3.21 mg in 100 µL water) on a C18 column (4.6×250 mm). Elution was carried out at 1 mL/min with a 0–60% ACN gradient. Eight fractions were collected (F4-a - F4-h). 3.- Reverse-Phase HPLC of F4-h fraction (0.63 mg in 100 µL water) on a C18 column using an 18–27% ACN gradient. Dotted lines indicate the ACN gradient used.

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Browplasminin was thus isolated employing three chromatographic steps: first, a size exclusion chromatography that allowed obtains the low molecular weight fraction (F4) with high anti-plasmin activity. Second, a separation based on polarity using a reverse phase column where the majority of the components require of 12 to 30 min (12–30% ACN gradient) to elute. This step allowed obtaining a low polarity fraction and with specificity for plasmin (F4-h). Similar results were obtained by Jin and Row (2007) who successfully separated five catechins: the (+) catechin, (-) epicatechin, (-) epigallocatechin gallate and (-) epigallocatechin, using a 10–30% ACN gradient. A third reverse phase step produced the purified Browplasminin.

3.2. MALDI-ToF analysis

Condensed tannins as well as other phenolic compounds can be studied using mass spectrometry techniques, such as MALDI-ToF which detects and allowed to characterize high molecular weight proanthocyanidins (Fulcrand et al., 2008). Browplasminin was analyzed by MALDI-ToF mass spectrometry and [M+Na]⁺ or [M+Cs]⁺ pseudo molecular ions were generated and detected separately in positive mode, while [M-H]^- pseudo molecular ions were obtained and studied in negative mode. The results showed that Browplasminin consists of predominating heteroflavan-3-ols of catechin with B-type linkages, and extend up to heptadecamers with hexose residues attached to the polymer that presents a high degree of galloylation and a molecular mass of approximately 5000 Da (Table 1, Fig. 2 and Fig. 3).

Table 1.. Comparison of MALDI-ToF MS mass spectra of Browplasminin in lineal positive-ion mode ([M+Na]⁺ and [M+Cs]⁺ adducts) and in lineal negative-ion mode ([M-H]^-adduct). This analysis revealed that Browplasminin was built up from a mixture of ofafzelechin, catechin and gallocatechin in different proportions to produce distinct polymers. The masses were calculated using the equation 290+288(a) + c, where 290 represents the molecular weight of the terminal catechin/epicatechin unit, 288 is the molecular weight of the extended catechin/epicatechin unit, a is the degree of polymerization and c is the weight of the adduct ion (23 for sodium,133 for Cesium and −1 for hydrogen). (-) mass not observed.

Polymer	Afzelechin	Catechin	Gallocatechin	[M + Na]+ calculated	[M + Na]+ detected	[M + Cs]+ calculated	[M + Cs]+ detected	[M - H]- calculated	[M - H]- detected
Dimer	1	1	0	585	585.0726	695	-	561	561.4493
	0	2	0	601	601.0977	711	-	577	577.4679
	0	1	1	617	617.1465	727	727.3595	593	593.4865
Trimer	1	2	0	873	873.6940	983	983.6000	849	-
	0	3	0	889	889.5397	999	999.5172	865	865.0340
	0	2	1	905	905.7588	1015	-	881	881.2339
	0	1	2	921	921.6294	1031	1031.4091	897	897.2843
Tetramer	1	0	3	1161	1161.7991	1271	1271.0157	1137	1137.4435
	0	4	0	1177	1177.7108	1287	1287.3306	1153	1153.4581
	0	3	1	1193	1193.8573	1303	1303.4537	1169	1169.3812
	0	2	2	1209	1209.7640	1319	-	1185	1185.5648
Pentamer	1	4	0	1449	1449.0116	1559	1559.2095	1425	1425.4473
	0	5	0	1465	1465.3619	1575	1575.2159	1441	1441.7540
	0	4	1	1481	1481.3157	1591	1591.4082	1457	1457.7330
Hexamer	1	5	0	1737	1737.2510	1847	1847.4553	1713	1713.2187
	0	6	0	1753	1753.2098	1863	1863.2400	1729	1729.1918
	0	5	1	1769	1769.2264	1879	1879.1970	1745	1745.1649
Heptamer	1	6	0	2025	2025.2556	2135	2135.3275	2001	2001.1533
	0	7	0	2041	2041.2424	2151	2151.3313	2017	2017.1539
	0	6	1	2057	2057.3288	2167	2167.3481	2033	2033.1182
Octamer	1	7	0	2313	2313.2548	2423	2423.6880	2289	2289.4788
	0	8	0	2329	2329.3163	2439	2439.3253	2305	2305.7157
	0	7	0	2345	2345.3376	2455	2455.3824	2321	23212445
Polymer	Afzelechin	Catechin	Gallocatechin	[M + Na]+ calculated	[M + Na]+ detected	[M + Cs]+ calculated	[M + Cs]+ detected	[M - H]- calculated	[M - H]- detected
Nonamer	1	8	0	2601	2601.7129	2711	2711.3730	2577	2577.8876
	0	9	0	2617	2617.3587	2727	2727.2340	2593	2593.9589
	0	8	1	2633	2633.3423	2743	2743.5238	2609	2609.8579
Decamer	1	9	0	2889	-	2999	-	2865	2865.5669
	0	10	0	2905	-	3015	3015.8587	2881	2881.3149
	0	9	1	2921	-	3031	3031.3059	2897	2897.2576
Undecamer	1	10	0	3177	-	3287	3287.0259	3153	3153.3707
	0	11	0	3193	-	3303	3303.1196	3169	3169.7681
	0	10	1	3209	-	3319	-	3186	3186.7018
Dodecamer	0	12	0	3481	-	3591	3591.7866	3457	3457.5681
Tridecamer	0	13	0	3769	-	3879	-	3745	3745.6782
Tetradecamer	0	14	0	4057	-	4167	-	4033	4033.3955
Pentadecamrer	0	15	0	4345	-	4455	-	4321	4321.7355
Hexadecamer	0	16	0	4633	-	4743	-	4609	4609.3887
Heptadecamer	0	17	0	4921	-	5031	-	4897	4897.4825

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Fig. 2. 

Browplasminin MALDI-ToF lineal mode mass spectra in the range m/z=800–5000. A) [M+Na]⁺ adducts that resulted from the addition of NaCl to the matrix/analyte solution. B) [M+Cs]⁺ ions from addition of CsCl. C) Negative-ion mode [M-H]^- signals. DP: degree of polymerization; g: galloylated monomer.

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Fig. 3. 

Details of the MALDI ToF mass spectra of Browplasminin. A) Amplification in the range m/z=1700–2100 of the MALDI ToF negative-ion mass spectra of Browplasminin, that shows signals at a distance of 152 Da corresponding to the presence of one galloyl group. Repetitive patterns of peaks are characterized by mass increments of 274 Da, 288 Da and 304 Da, corresponding to afzelechin, catechin and gallocatechin repeating units, respectively. The 16 Da mass difference is related to a hydroxyl group. B) Amplification of the m/z=855–910 region of the positive-ion NaCl MALDI ToF mass spectrum that shows signals that are 2 Da lower than the predicted peaks for heteropolyflavan-3-ols. C) Expansion of the range m/z=900–1600 of positive-ion CsCl MALDI ToF mass spectrum showing peaks followed by mass signals at a distance of 162 Da. These masses correspond to [M+H]⁺ ions that were also detected.

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The spectra, summarized in Fig. 2, were characterized by repetitive patterns of peaks with a distance of 288 Da between each set that corresponded to catechin/epicatechin monomeric units (Khanbabaee and van Ree, 2001), the addition of which produced polymeric molecules. Thereby, an equation was developed to predict the mass distribution of polyflavans using the terms: 290+288(a) + c, where 290 represents the molecular weight of the terminal catechin/epicatechin unit, 288 is the molecular weight of the extended catechin/epicatechin unit, the alphabetic character a, represents the degree of polymerization, while alphabetic character c, is the weight of the adduct ion employed (23 for sodium, 133 for Cesium and −1 for hydrogen). So, spectra analysis provided detection of oligomeric sodium adducts that extended up to nonamers (m/z=2617.3587) and of cesium adducts up to dodecamers (m/z=3591.7866) (Table 1). Negative-ion mode allowed more resolution and the detection of masses that correspond to an oligomeric series of catechin/epicatechins up to the heptadecamers (m/z=4897.4825) (Table 1).

Fig. 3A shows that each periodic peak series presented signals 16 Da higher that might be related to the presence of an additional hydroxyl group at the position 5' of the B-ring. Furthermore, it was possible to detect signals 16 Da lower that might the indicated monomers that contain only one hydroxyl group attached to B aromatic moiety. Thus, it was suggested that Browplasminin contains flavan-3-ols type catechin/epicatechins, gallocatechin/epigallocatechins and afzelechin/epiafzelechins, meaning, procyanidins, prodelphinidins and propelargonidins, respectively (Khanbabaee and van Ree, 2001).

The mass spectra also showed that the signals of catechin/epicatechin oligomers are more intense with respect to the gallocatechin/epigallocatechin and afzelechin/epiafzelechin peaks series, indicating that procyanidins were the main constituents of Browplasminin. The masses of sodium adduct ions [M+Na]⁺ obtained in this work had the same values as in Krueger et al. (2000), who attributed them to the presence of procyanidins, prodelphinidins and properlagonidin polymers in tannins from grape seeds (Krueger et al., 2000 and Reed et al., 2005), leaves and needles from Salix alba, Picea abies, Fagus sylvatica and Tilia cordata ( Behrens et al., 2003) and oligomeric procyanidin fractions from grape seeds and condensed tannin fractions from apple (Mané et al., 2007). Whereas the masses of cesium adducts ions [M+Cs]⁺ detected here agree with the masses observed for tannins from Canarium album ( Zhang and Lin, 2008), Kandelia candel and Rhizosphora mangle leaves ( Zhang et al., 2010a), Casuarina equisetifolia stem bark and fine roots ( Zhang et al., 2010b), and procyanidins and prodelphinidins from Grevillea robusta leaves ( Wei et al., 2012).

Moreover, the spectra contained masses that are 2 Da lower than those already described (Fig. 3B) that could be attributed to A-type interflavanic linkages that occur between flavan-3-ol subunits (Wei et al., 2011 and Flamini, 2013). These peaks showed lower intensity with respect to the corresponding compounds with B-type bonds, thus implying that, in Browplasminin there are links of B-type and A-type bonds, with the B-type linkages predominating. MALDI-ToF MS has revealed the presence of A-type interflavan bonds in condensed tannins isolated from cranberry (Foo et al., 2000), Tilia cordata ( Behrens et al., 2003), and Machilus pauhoi ( Wei et al., 2011).

In all spectra, the periodical occurrence of sets of peaks with mass differences of 152 Da was detected (Fig. 2, Fig. 3A). These signals correspond to one galloyl ester residue at the heterocyclic C-ring (Flamini, 2013). The negative mode is the best analytical condition for the detection of galloylated structures, followed by spectra performed in positive mode using CsCl, whereas poor signals are expected in analysis carried out with NaCl.

To predict the mass distribution of galloylated polyflavonoids, the equation used was the one described by Da Silva et al. (1992): 290 + 288a+ 152b + c, where 290 is the molecular weight of the terminal catechin/epicatechin unit, 288 is the molecular weight of the extended catechin/epicatechin unit, a represents the degree of polymerization, 152 represents the galloyl group, b is the degree of galloylation, c symbolizes the weight of the adduct ion employed (23 for sodium,133 for Cesium and −1 for hydrogen). The occurrence of galloyl residues on some of the repeating catechin/epicatechin units is one of the most common modifications in proanthocyanidins and has been reported for tannins isolated from oligomeric procyanidin fractions from grape seeds and condensed tannin fractions from apple (Krueger et al., 2000 and Mané et al., 2007), Canarium album plants ( Zhang and Lin, 2008) and Zyzygium cumini fruit ( Zhang and Lin, 2009).

Polyflavonoid peaks were followed by signals separated by 162 Da (Fig. 3C) which can be related to the presence of one glycoside group at the heterocyclic C ring, suggesting that Browplasminin is glycosylated (Zhang et al., 2010a). The glycosylated forms were detected in negative mode and spectra with [M+Cs]⁺ adducts. Fig. 3B displays an amplification of the spectrum with Cs⁺with details of the peaks; [M+H]⁺ adducts were observed with high intensity until a region of m/z=1500. Glycosylation is another important modification on flavonoids and has been detected in condensed tannins from Kandelia candel and Rhizosphora mangle ( Zhang et al., 2010a).

In brief, using three methods of spectrum acquisition for MALDI-ToF MS analysis it was possible to determine the main characteristics and components of the condensed tannin. The best analytical condition was the negative-ion mode which is consistent with the weak acidic nature of the procyanidins, where the proton dissociation in negative mode is much more feasible than protonation in positive-ion mode (Flamini and Traldi, 2010).

3.3. Plasmin inhibition

The results demonstrated that BGE, F4, F4-h, Browplasminin, aprotinin and TA at 100 µg/mL of final concentration, abolished the plasmin activity in fibrin plates, while EACA had a lesser activity (Table 2). Besides, when assayed, the effect of the extract, fractions and Browplasminin at a 100 µg/mL of final concentration on the amidolytic activity of plasmin was observed that Browplasminin inhibited the plasmin activity by 94.97%, whereas aprotinin, TA and EACA diminished the activity by 99.01, 81.70, and 20.72%, respectively (Table 2). These results corroborate those reported by us when we previously studied the BGE ( Pereira and Brazón, 2015). On the other hand, using the amidolytic method, it was observed that Browplasminin increases the inhibitory activity on plasmin in a dose-dependent manner (Fig. 4).

Table 2.. Anti-fibrinolytic activity of BGE and its fractions on fibrin plates and amidolytic activity. Plasmin (0.652 µM) was pretreated with extract or fractions (100 μg/mL) for 30 min at 37 °C. Ten microliters of the plasmin/sample mix described above were placed on a fibrin plate and incubated at 37 °C for 24 hours. Lytic areas were measured in mm² and the inhibitory activity was expressed in percentage. Also, other ten microliters of the plasmin/sample mix were allowed to react with the chromogenic substrate S-2251. The amidolytic activity was measured at 405 nm and the results are shown as the median and their CI at 90% confidence, n=3. Percentage of inhibitory activity was calculated. Aprotinin, EACA and TA (100 μg/mL) were used as reference inhibitors.

Sample	Fibrin lysis (mm2)	Inhibitory activity (%)	Amidolytic activity (UA/min/µg)	Inhibitory activity (%)
Plasmin (Pln)	340.5	0	357.67 (230.67–430.67)	0
Pln + BGE	0	100	40.77 (20.88–69.88)	88.99
Pln + F4	0	100	47.89 (43.44–52.44)	86.61
Pln + F4-h	0	100	33.00 (29.89–40.00)	90.77
Pln + Browplasminin	0	100	18.00 (17.67 − 23.33)	94.97
Pln + Aprotinin	0	100	3.56 (3.33–4.00)	99.01
Pln + EACA	250	26.58	283.56 (283.33–288.67)	20.72
Pln + TA	0	100	65.44 (65.00–74.22)	81.70

Table options

Fig. 4. 

Inhibitory effect of Browplasminin on the amidolytic activity of plasmin and Factor Xa. The inhibitory activity was determined incubating each enzyme with different concentrations of Browplasminin (6.25, 12.5, 25, 50 and 100 µg/mL) for 30 min at 37 °C. The residual activity of plasmin (0.652 µM) or FXa (0.7 µM) was evaluated measuring p-nitroaniline liberation at 405 nm from the chromogenic substrate specific to each one. The results are shown as % of inhibition from the median (UA/min/μg) and their CI at 90% confidence, n=3. The IC₅₀ values were determined employing a logarithmic non-linear regression analysis and is indicated here. ■ Plasmin pretreated with Browplasminin. ● FXa pretreated with Browplasminin.

Figure options

The inhibitory effect of Browplasminin, a condensed tannin of approximately 5 kDa, has showed to be higher than the reference compounds EACA (131 Da) and TA (157 Da) but similar in molecular mass and inhibitory effect to aprotinin (Table 3). This find could help explain how the (anti-plasmin) anti-fibrinolytic activity of Browplasminin could be related to the reduction of blood loss reported by women that practice the Venezuelan traditional medicine (Díaz and Ortega, 2006 and Pereira and Brazón, 2015).

Table 3.. Effect of BGE and its fractions on amidolytic activity of factor Xa (FXa). The coagulation factor (0.7 μM) was incubated with BGE or fractions (100 μg/mL) for 30 min at 37 °C. Ten microliters of this mix were incubated with the chromogenic substrate S-2222. The activity was measured at 405 nm and the results are shown as the median and their CI at 90% confidence, n=3. Percentage of inhibitory activity was calculated. Benzamidine (10 mM) was used as reference inhibitors.

Sample	Amidolytic activity (UA/min/µg)	Inhibitory activity (%)
Factor Xa (FXa)	339.00 (302.33 − 388.55)	0
FXa + BGE	90.89 (88.44 − 175.33)	73.19
FXa + F4	89.55 (88.00 − 114.22)	73.58
FXa + F4-h	88.21 (68.89–90.11)	73.98
FXa + Browplasminin	87.22 (69.66 − 99.87)	74.27
FXa + Benzamidine	8.44 (7.89 − 10.22)	97.51

Table options

This work is the first study that shows anti-plasmin activity in condensed tannins. The inhibitory activity of these compounds has been mostly characterized on digestive enzymes (Tamir and Alumot, 1969; Gonçalves et al., 2007; 2011), protein kinases (Wang et al., 1996), DNA topoisomerase II (Kashiwada et al., 1993), HIV-1 protease (Ma et al., 2000); fatty acid synthase (Zhang et al., 2008) and tyrosinases (Chai et al., 2012 and Chen et al., 2014). Nevertheless, plasmin inhibition has been described in flavonoids isolated from plants like the Blumea balsamifera ( Osaki et al., 2005, Mozzicafreddo et al., 2008 and Cuccioloni et al., 2009).

3.4. Inhibitory activity of Browplasminin on others serine-proteases

In order to determine the specificity of Browplasminin, its inhibitory activity was screened against serine-proteases in the coagulation and fibrinolytic systems using their respective chromogenic substrates. The results showed that Browplasminin had no significant effect on amidolytic activities of trypsin, t-PA or u-PA (data not shown). However, Browplasminin diminished FXa amidolytic activity in a dose-dependent manner, yet its effect was more efficient on plasmin (Fig. 4). In 2015 we reported similar findings when studying BGE activity on t-PA, u-PA, FXa and plasmin at concentrations ≤100 µg/mL (Pereira and Brazón, 2015).

It has been reported that some flavonoids are capable of inhibiting serine proteases related to haemostasis such as epigallocatechin gallate and tannic acid that repress u-PA, plasminogen activator inhibitor and elastase (Jedinak and Maliar, 2005, Cuccioloni et al., 2009 and Cale et al., 2010), quercetin which has inhibitory effects on trypsin, thrombin and u-PA (Jedinak et al., 2006 and Danihelová et al., 2013); delphinidin, cyanidin, pelargonidin and fisetinidin that abolishes the activating protease of Factor VII (Yamamoto et al., 2011).

3.5. Kinetic characteristics of Browplasminin

The IC₅₀ for plasmin and FXa were estimated through a non-linear regression analysis (Fig. 4). Browplasminin inhibited 50% of plasmin activity at concentration of 47.80 μg/mL, while the IC₅₀ for FXa is 237.08 μg/mL. The inhibition constants (Ki) deduced from Eq. (2) were 0.76 and 61.61 μg/mL for plasmin and FXa, respectively. The results indicated that the Browplasminin inhibition is more specific for plasmin. Condensed tannins with similar structural characteristics to Browplasminin exhibited IC₅₀ values between 74 and 128 μg/mL (Mozzicafreddo et al., 2008 and Chen et al., 2014); this evidence suggests that Browplasminin could be considered a good plasmin inhibitor.

A relevant structural detail of Browplasminin is its high degree of galloylation (more hydroxyl groups in its structure), which allow more interaction of these hydroxyl groups through H-bonds with the active site of the enzyme (Mozzicafreddo et al., 2006; Cuccioloni et al., 2009; Chen et al., 2014), this suggests that Browplasminin could have a facilitated access to the catalytic pocket of the plasmin molecule in order to produce the inhibitory effect. In fact, Osaki et al. (2005) demonstrated that flavonoids from Blumea balsamifera with hydroxyl groups on 3' and 4' positions had a potent anti-plasmin effect. Thus, it is possible that a high content of galloyl residues in Browplasminin is related to its inhibitory efficiency. Nevertheless, more studies are needed to determine the type of inhibitor-enzyme interactions and if the galloylation of Browplasminin is involved in the mechanism of plasmin inhibition.

4. Conclusions

The BGE is a rich source of inhibitory compounds, principally phenolic molecules of a broad range of molecular weights with different efficiencies to inhibit plasmin and FXa. A condensed tannin isolated from BGE, which has been called Browplasminin, mainly showed anti-plasmin activity. Through MALDI-ToF mass spectrometry analysis it was determined that Browplasminin contains predominantly catechins of B-type which form a tannin polymer about 5000 Da that is highly galloylated and presents some residues of hexose. The inhibitory activity on plasmin (IC₅₀=47.80 μg/mL, Ki = 0.76 μg/mL) of Browplasminin could be related to its high degree of galloylation, however, further research is required to determine its plasmin inhibition mechanism.

Statement of authors' contributions to manuscript

B.P (bpereira@ivic.gob.ve) and J.B (jbrazon@ivic.gob.ve) conducted research, analyzed data and wrote the paper. J.B designed research. M.R (monicarincong@gmail.com) and E.V (evonasek@ivic.gob.ve) conducted the assay with MALDI-ToF SM. All authors read and approved the final manuscript. The authors declare no conflict of interests.

Acknowledgments

The authors are indebted to the family Baasch Castrillo of Posada Mocundo, Carabobo State, Venezuela for the supply of Brownea grandiceps flowers. We greatly appreciated the technical assistance of Moises Sandoval, Felix Castillo and Milagros Mendez. This research was supported by grants from the Fondo Nacional de Ciencias, Técnología e Innovación-Programa Estímulo a la Innovación e Investigación (FONACIT-PEII) N° 2012001444 and to the Instituto Venezolano de Investigaciones Científicas (IVIC).

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Correspondence to: Laboratorio de Neurofarmacología Celular, Centro de Biofísica y Bioquímica, IVIC. Apartado 20632, Caracas 1020-A, Venezuela. Tel.: +58 212 504 1479; fax: +58 212 504 1093.

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