Anticoagulant activities of piperlonguminine in vitro and in vivo

Abstract

Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit anti-hyperlipidemic, anti-platelet and anti-melanogenic activities. Here, the anticoagulant activities of PL were examined by monitoring activated-partial-thromboplastin-time (aPTT), prothrombin-time (PT), and the activities of thrombin and activated factor X (FXa). The effects of PL on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated HUVECs. The results showed that PL prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. PL inhibited the generation of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, PL prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significan- tly decreased by PL. Collectively, our results suggest that PL possesses antithrombotic activities and that the current study could provide bases for the development of new anticoagulant agents.

Fig. 1.. Structure and characterization of piperlonguminine (PL) and effects of PL on fibrin polymerization in human plasma. (A) Structure and HPLC chromatogram of PL. (B) Effect of PL on mouse platelet aggregation induced by 3 U/ml thrombin. (C) Thrombin (Th, white box) or reptilase (Rep, black box) catalyzed fibrin polymerization by the indicated concentrations of PL was monitored using the catalytic assay described in “Materials and Methods”. (D) Effect of PL on cellular viability was measured by MTT assay. Data represent the means ± SDs of 3 independent experiments performed in triplicate. The results are Vmax values expressed as percentages of thrombin alone. *P ＜ 0.05 or **P ＜ 0.01 as compared to Th alone (B) or Th/Rep alone (C).

Fig. 2.. Effects of PL on the inactivation and on the generation of thrombin and factor Xa. (A) Inhibitions of thrombin or (B) factor Xa by PL. (C) Generation of thrombin or (D) factor Xa in HUVECs. Heparin (40 μg/ml) was used as positive control. *P ＜ 0.05 or **P ＜ 0.01 as compared to 0 (A-C) or TNF-α alone (D).

Fig. 3.. Effects of PL on the secretion of PAI-1 and t-PA. (A) HUVECs were cultured with PL in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentrations in the culture media were measured. (B) HUVECs were cultured with PL in the absence or presence of TNF-α (10 ng/ml) for 18 h and t-PA concentrations in culture media were measured. (C) PAI-1/t-PA ratio in TNF-α activated HUVECs from (A) and (B). **P ＜ 0.01 as compared to TNF-α alone (A, B) or 0.; n.s., not significant.