Aqueous extract from Brownea grandiceps flowers with effect on coagulation and fibrinolytic system

Betzabeth Pereira,
Josmary Brazón^,^,

doi:10.1016/j.jep.2014.11.022

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Abstract

Ethnopharmacological relevance

Brownea grandiceps flowers are used in Venezuelan folk medicine as anti-hemorrhagic in women with heavy menstrual blood loss (menorrhagia). However, prior to this study, there were no scientific investigations to support this fact, because the aqueous extract from Brownea grandiceps flowers had not been previously evaluated neither phytochemically nor biologically. The objective of this work was to evaluate in vitro the effects of aqueous extract from Brownea grandiceps flowers on the coagulation system and fibrinolysis.

Materials and methods

An infusion of Brownea grandiceps flowers (160 g) was performed; then, it was homogenized, centrifuged and lyophilized to obtain the aqueous extract, and this was called BGE. Subsequently, the extract was characterized on the one hand, phytochemically and on the other hand, biologically, employing prothrombin time (PT), partial thromboplastin time (PTT) and thrombin time (TT) to determine the effects on extrinsic, intrinsic and common coagulation pathways, respectively. In addition to that, the fibrinogenolytic and fibronectinase activity was evaluated by SDS-PAGE using Tris-Tricine system and analyzed by densitometric study utilizing ImageJ program. Also, by using specific chromogenic substrates for Factor Xa (FXa), thrombin, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasmin, it was assessed whether BGE exhibited some enzyme-like activity, and inhibitory activity of the afore mentioned enzymes. Fibrinolytic and antifibrinolytic activities were determined by a fibrin plate method. Data were analyzed by an nonparametric method.

Results

BGE presented tannins, saponins, glycosides, alkaloids, flavonoids, coumarins, and did not contain triterpenoids and steroids. Also, BGE at low concentrations (250–1250 µg/mL) reduced the PT, while higher concentrations (15000–25000 µg/mL) prolonged this time. However, BGE concentrations between 1250 and 25000 µg/mL prolonged the PTT. Prolongation of PT and PTT was observed at high concentrations and was due to FXa inhibitor found in BGE and this effect could be strengthened by degradation of fibrinogen and fibronectin, which were also produced by BGE. Moreover, BGE did not clot fibrinogen or human plasma, and neither did it cleave the chromogenic substrates specific to FXa nor thrombin. These results suggest the pro-coagulant components could be acting on some factor of the extrinsic pathway, since only PT was shortened. Furthermore, BGE did not hydrolyze the chromogenic substrate specific to plasmin, t-PA and u-PA nor did it produce fibrin degradation. However, all BGE concentrations tested inhibited the plasmin activity in a dose-dependent manner.

Conclusions

The outcomes of this study reveal the presence of fibrinogenolytic, fibronectinase and anti-FXa components in BGE, plus anti-plasmin compounds that could be acting as antifibrinolytic, thus delaying the fibrin degradation in pathophysiological processes, as it has been observed in women presenting with menorrhagia due to a high plasmin concentration. Where this anti-plasmin compound, along with pro-coagulant components also present in BGE, could be made responsible for reducing heavy menstrual bleeding in women, since a deficiency in one or more blood coagulation factors such as factor VII, V or X, is a potential cause of menorrhagia.

Graphical abstract

Abbreviations

BGE, Brownea grandiceps flowers aqueous extract;
CI, confidence interval;
FXa, Factor Xa;
Fg, fibrinogen;
FgC, fibrinogen control;
FN, fibronectin;
FNC, fibronectin control;
g, grams;
g, gravity;
h, hour;
L, liters;
m, meters;
min, minutes;
MW, molecular weight;
PPP, platelet poor plasma;
PT, Prothrombin time;
PTT, Partial thromboplastin time;
s, seconds;
SDS-PAGE, Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis;
t-PA, tissue plasminogen activator;
u-PA, urokinase plasminogen activator.

Keywords

Brownea;
Antiplasmin;
Fibrinogen;
Anti-coagulant;
Fibronectin;
Pro-coagulant

: Corresponding author. Tel.: +58 212 504 1479; fax: +58 212 504 1093.

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Monday, 6 April 2015