Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode,Heligmosomoides polygyrus,in vitro

Abstract

A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD 50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris

“…2A-C, papaya latex, fig latex and pineapple fruit extract (containing active cysteine proteinases) caused progressive damage to the cuticle, beginning with the appearance of transverse wrinkles, followed by blistering and, finally, less localized cuticle digestion with expulsion of the worm intestine and other internal structures from points along the worm surface. Although these enzymes displayed a similar effect with T. muris to that previously documented with H. polygyrus (Stepek et al 2005), the damage took slightly longer to occur.…”

“…This study has demonstrated conclusively that the anthelmintic efficacy of various plant cysteine proteinases observed in our previous study with Heligmosomoides polygyrus (Stepek et al 2005) is not species-specific, as these enzymes are comparably effective against the adult worms of Trichuris muris, a parasite that occupies the large intestine, predominantly living in the mouse caecum. Adult worms of this species were affected during in vitro incubation in the presence of most of the plant cysteine proteinases tested.…”

“…In order to standardize all enzyme preparations, the operational molar concentration of active cysteine proteinase was determined by active-site titration with L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), as described previously (Stepek et al 2005).…”

“…The effects of the cysteine proteinases on the motility of the adult male and female worms were determined following the method described previously by Stepek et al (2005) for Heligmosomoides polygyrus, using the following concentrations of active enzymes : 0-100 mM (25 mM increments) papain, 0-200 mM (50 mM increments) chymopapain, 0-200 mM (50 mM increments) crude papaya latex proteinase, 0-300 mM (50 mM increments) F. carica latex proteinase, 0-500 mM (100 mM increments) F. benjamina latex proteinase, 0-50 mM (10 mM increments) ficin, 0-150 mM (50 mM increments) stem bromelain, 0-200 mM (100 mM increments) fruit bromelain and 0-300 mM (100 mM increments) actinidain. At the same time, adult male and female worms were incubated with either 200 mM crude papaya latex or 150 mM stem bromelain after inhibition of the cysteine proteinases in both preparations by the addition of 3 mM E-64.…”

The anthelmintic efficacy of plant-derived cysteine proteinases against the rodent gastrointestinal nematode,Heligmosomoides polygyrus,in vivo

“…However, to our knowledge, the current study is the first (i) to establish, unequivocally, that it is upon the cysteine proteinases present in the latex that the anthelmintic efficacy of papaya latex depends, and (ii) to provide evidence that the mechanisms of action in vivo and in vitro are essentially indistinguishable. In vitro studies (Stepek et al 2005(Stepek et al , 2006(Stepek et al , 2007 have demonstrated that cysteine proteinases attack the protective cuticle of the nematode, rapidly leading to complete dissolution of the parasite, a mechanism of action that differs from all the current anthelmintic drugs (Sangster and Gill, 1999 ;Martin and Robertson, 2000). This effect was similar to that described previously for 3 other gastrointestinal nematodes, H. polygyrus (Stepek et al 2005), T. muris (Stepek et al 2006) and Ascaris suum (Robbins, 1930 ;Berger and Asenjo, 1939), indicating that plant cysteine proteinases have a broad spectrum of activity against GI nematodes in vitro.…”