Structural Properties of Macrodontain I, a Cysteine Protease from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman's degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M-1 cm-1. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α + ß class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the ß domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.

Effects on fibrinogen, fibrin, and blood coagulation of proteolytic extracts from fruits of Pseudananas macrodontes, Bromelia balansae, and B. hieronymi (Bromeliaceae) in comparison with bromelain.

Extracts rich in cysteine proteases obtained from fruits of Pseudananas macrodontes (Pm), Bromelia balansae (Bb), and B. hieronymi (Bh) have previously shown an anti-inflammatory effect on animal models. Given the close relationship between hemostasis and inflammation, it is attractive to investigate therapeutic agents capable of modulating both systems. The aim of this work was to study the effect of Pm, Bb, and Bh on fibrin(ogen) and blood coagulation compared with stem bromelain (Bro). Action on fibrinogen was electrophoretically and spectrophotometrically evaluated, fibrinolytic activity was measured both electrophoretically and by the fibrin plate assay, and the effect on blood coagulation was studied by conventional coagulation tests (PT and APPT). All extracts showed the same proteolytic preference for fibrinogen subunits, that is Aα > Bß, whereas γ was partially hydrolyzed by 100-fold concentration increase. Unlike Bro, cysteine proteases of Pm, Bb, and Bh increased absorbance at 540 nm of fibrinogen solution, suggesting thrombin-like activity, which was time-dependent and reached maximum values at lower concentration. All extracts showed the same proteolytic preference for fibrin subunits; however Pm, Bb, and Bh showed lower fibrinolytic activity than Bro at the assayed concentrations. Although Bb acted only as anticoagulant, Pm, Bh, and unexpectedly Bro showed dual action on blood coagulation: at low concentration showed procoagulant effect and at high concentration anticoagulant effect. Results reveal new plant species as potential sources of pharmacological agents for the treatment of a wide range of hemostatic disorders as well as to wound healing.

Anti-inflammatory activity of Bromelia hieronymi: comparison with bromelain.

Some plant proteases (e. g., papain, bromelain, ficin) have been used as anti-inflammatory agents for some years, and especially bromelain is still being used as alternative and/or complementary therapy to glucocorticoids, nonsteroidal antirheumatics, and immunomodulators. Bromelain is an extract rich in cysteine endopeptidases obtained from Ananas comosus. In this study the anti-inflammatory action of a partially purified extract of Bromelia hieronymi fruits, whose main components are cysteine endopeptidases, is presented. Different doses of a partially purified extract of B. hieronymi were assayed on carrageenan-induced and serotonine-induced rat paw edema, as well as in cotton pellet granuloma model. Doses with equal proteolytic activity of the partially purified extract and bromelain showed significantly similar anti-inflammatory responses. Treatment of the partially purified extract and bromelain with E-64 provoked loss of anti-inflammatory activity on carrageenan-induced paw edema, a fact which is consistent with the hypothesis that the proteolytic activity would be responsible for the anti-inflammatory action.

Evaluation of anti-inflammatory activity of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae) fruit extract in rats.

Several species of the family Bromeliaceae are characterized by the production of proteases in unusual amounts, especially in fruits. Bromelain, an extract rich in cysteine endopeptidases obtained from Ananas comosus L., and a few other proteases have been used as anti-inflammatory agents for some years, but bromelain is still mainly being used as alternative and/or complementary therapy to the treatment with glucocorticoids, nonsteroidal antirheumatics, and immunomodulators. In this study, the anti-inflammatory action of a partially purified extract from Pseudananas macrodontes (Morr.) Harms fruits (PPE(Pm)) is presented, whose main components are cysteine endopeptidases. The effect of PPE(Pm) was assessed in carrageenan-induced and serotonin-induced rat paw edema, as well as in the cotton pellet granuloma model. Doses with equal proteolytic activity of PPE(Pm) and bromelain produced significantly similar anti-inflammatory responses in the acute inflammatory models assayed, supporting the hypothesis that proteolytic activity could be responsible for the anti-inflammatory action. On the contrary, comparable anti-inflammatory effects of PPE(Pm) and bromelain in the chronic inflammatory assay required a much lower proteolytic activity content of PPE(Pm), which could be due to a differential affinity for the protein target involved in this process.