Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

Abstract

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera.

Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug

“…1b, the highest protease inhibitor activity was recorded in the crude extract prepared in 0.1 M phosphate buffer (pH 7.6) (86 ± 5.1% inhibition) followed by the one prepared in distilled water (55 ± 6.1% inhibition). Similar results are reported for the maximal extraction of proteins from Moringa oleifera leaves (Bijina et al 2011) and Cajanus cajan seeds (Pichare and Kachole 1996). Thus, R. frangula leaves which may be a rich source of protease inhibitor were considered for further studies.…”

“…3c). Similar results were described for almost all plant protease inhibitors of Kunitz type of serine protease inhibitor family (Hamato et al 1995;Bijina et al 2011).…”

“…Furthermore, the effect of two reducing agents on the activity of protease inhibitor was also evaluated, and data were shown in Table 2, and it seems that both DTT and b-ME could enhance inhibitory activity up to 55% at 1% concentration (Table 2). Similar results were reported for the protease inhibitor from M. oleifera (Bijina et al 2011) which suggested that, in the presence of reducing agents, the functional stability of Kunitz-type protease inhibitors is presumably attributed to the intramolecular disulphide (Kunitz 1947).…”

“…(10 mM) markedly enhanced the activity of the protease inhibitor purified from Moringa oleifera leaves (Bijina et al 2011). Data obtained in the present study suggested that metal ions bind to the R. frangula protease inhibitor and stabilize it spatial conformation.…”

“…3b). Several protease inhibitors in the Kunitz family were previously reported to be stable in the alkaline pH, while they are very sensitive to acidic pH losing their full activity when incubated at pH values of less than 5 for few minutes (Mello et al 2002;Bijina et al 2011). Kridric et al suggested that the functional stability of Kunitz-type protease inhibitors in the extreme conditions of pH and temperature and when incubated with reducing agents is probably due to the intra molecular disulphide bridges (Kridric et al 2002).…”