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Vol.20 No.7 July 2004

Natural plant cysteine proteinases as anthelmintics?

Gillian Stepek1, Jerzy M. Behnke1, David J. Buttle2 and Ian R. Duce1
School of Biology, University Park, University of Nottingham, Nottinghamshire, NG7 2RD, UK Division of Genomic Medicine, University of Shefeld, D-Floor, Stephenson Wing, Childrens Hospital, South Yorkshire, S10 2TH, UK
2 1

Infections with gastrointestinal nematodes have severe consequences for the health of millions of people worldwide, and cause serious economic losses in livestock farming. Current control relies heavily on anthelmintic drugs, to which resistance is now developing rapidly. Plant cysteine proteinases, from the fruits or latex of plants such as papaya, pineapple and g, have high proteolytic activities that are known to digest nematode cuticles, have low toxicity and have been used in traditional medicines against gastrointestinal nematodes for decades. These proteinases constitute strong candidates for a much needed alternative strategy for the treatment of gastrointestinal nematode infections of both humans and animals. Gastrointestinal (GI) nematodes, also referred to as soiltransmitted nematodes, are parasites of major importance throughout the world because of the economic damage and suffering that these infections cause in humans and domestic animals [1 4]. GI nematodes, such as whipworms, hookworms and the roundworm Ascaris lumbricoides, infect millions of people, especially in tropical regions where children under 15 years of age are the most affected [2,3,5,6]. Chan estimated that in 1990, on a global scale, 39 million disability-adjusted years (DALYs) were lost to GI nematode infections of humans, more than that attributed to malaria (35.7 million DALYs), measles (34.1 million DALYs) and motor vehicle accidents (31.7 million DALYs). Similarly, livestock agriculture, notably sheep and cattle farming worldwide, is subject to severe economic losses from GI nematode infections, as a consequence of the serious diseases associated with infection, for example, anemia in small ruminants infected with Haemonchus contortus, and parasitic gastro-enteritis from Ostertagia ostertagi, Teladorsagia circumcincta and Trichostrongylus spp. A recent survey investigating the priorities for health of livestock among smallholder livestock keepers in Africa and Asia identied GI nematode infections as their top concern [4]. Problems with current control strategies Several control strategies are available in the ght against GI nematodes of both domestic animals and humans (Box 1), but anthelmintic drugs are the most widely used
Corresponding author: Jerzy M. Behnke (jerzy.behnke@nottingham.ac.uk).

and they have had a major positive impact on human and animal health. Three classes of modern synthetic anthelmintics are recognized, each having a different mode of action: (i) group 1, the benzimidazoles (e.g. albendazole) which disrupt b-tubulin; (ii) group 2, the imidazothiazoles and/or tetrahydropyrimidines (e.g. levamisole) which act on nicotinic acetylcholine receptors; and (iii) group 3, the macrocyclic lactones (e.g. ivermectin) which open glutamate-gated chloride channels. However, anthelmintics are not without problems. Ivermectin is excreted in the faeces in sufcient quantity to have a detrimental effect on invertebrates that normally degrade dung heaps, and thence on organisms higher up the food chain [7]. Growing consumer concern about potential synthetic drug residues in animal products is another problem [8,9]. Anthelmintics are relatively expensive for smallholder farmers in

Box 1. Strategies for achieving effective control of gastrointestinal nematode infections

To achieve effective control of gastrointestinal (GI) nematode infections in humans and livestock, a combination of different control strategies is required. For infections in livestock, good grazing management (such as eld rotation or rotating between different animal species, in addition to mixed ock or herd grazing), drug rotation between the different anthelmintic classes, and educating farmers on the most effective use of the anthelmintics would reduce the incidence of infections substantially and delay the onset of anthelmintic resistance. For infections in humans, improving hygiene and health care (by improving health education and awareness), ensuring patient compliance with treatment and drug rotation all have major roles to play in controlling and reducing infections with GI nematodes. However, this integrated control strategy is faced with numerous problems, the major ones being that: (i) chemotherapeutic drugs are either relatively cheap and so are overused, or are too expensive for the poorer farmers and communities hence under-dosing is common [11] but, either way, resistance of the nematodes to the drugs occurs; (ii) good grazing management requires ample land and/or different animal species for rotation, which is expensive; (iii) good sanitation and health care is often lacking in the worst infected areas because of the expense; (iv) farmers and the public might be ignorant about drug resistance and control methods, which can lead to a worsening situation; and (v) current treatment is limited by non-completion, sometimes as a result of unpleasant side-effects. No vaccines have yet been developed to a commercially viable stage and commercially available vaccines are unlikely to be available in the near future against GI nematodes of livestock and humans.

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relation to available resources, reducing their usefulness in developing regions of Africa and Asia [4,10]. Perhaps the most important problem arising from the increasing use of anthelmintics is the onset of resistance [11], analogous to crises affecting the use of antibiotics and insecticides. Resistance developed rst to group 1 anthelmintics in pastoral regions among species such as H. contortus, T. circumcincta and Trichostrongylus spp. [12], and then to the group 2 anthelmintics. More recently, triple resistance to all three groups of anthelmintics has developed among GI nematodes on goat farms in the UK, where the predominant parasite is T. circumcincta, and these triple-resistant nematodes have also infected sheep grazing concurrently on the same pasture [13 16]. Anthelmintic resistance in GI nematodes of livestock is most widespread throughout Australia, New Zealand, South America and South Africa, where ovine GI nematodes can no longer be treated satisfactorily by any anthelmintics because of resistance [13,17,18]. Anthelmintic resistance in GI nematodes of humans is poorly documented and probably still rare but, inevitably, regular use of anthelmintics will lead to resistance if control depends entirely on this strategy. For example, mebendazole resistance against the pinworm Enterobius vermicularis is already emerging in the UK [19]. There is evidence that mebendazole is losing efcacy against the hookworm Necator americanus in parts of Africa [20], and pyrantel is no longer effective against the hookworm Ancylostoma duodenale in Australia [21]. Thus, in view of the increasing problems associated with the use of current anthelmintic drugs, there is an urgent need for the development of alternative treatments against GI nematodes of both humans and livestock. The use of alternative control methods A potential alternative anthelmintic strategy is the use of extracts from medicinal plants [22,23]. These include many species of g tree from the genus Ficus and the papaya tree, Carica papaya, from which the crude latex was extracted and used successfully against ascarids, tapeworms, whipworms and hookworms in the early 19th century [24]. However, with the development of synthetic antiparasite drugs, use of natural plant remedies declined. Today, in the face of widespread resistance to synthetic drugs among parasitic nematodes, it is timely to re-examine the efcacy of some of these traditional treatments and investigate ways in which they can be improved [25]. Sources and modes of action A wide range of plants and plant extracts has been used traditionally for the treatment of helminth infections [22,26 29], including papaya, g and pineapple (Ananas comosus) [30]. Papaya and g trees produce latex upon injury (Figure 1a), which is rich in proteolytic enzymes, whereas other plants, such as the pineapple, contain large amounts of cysteine proteinases in the juices extracted from the stem or fruit [31] (up to 1 g of enzyme per pineapple on average, J.M. Behnke, unpublished). These enzymes are already in use in medicine, for example, chymopapain, which is used to treat prolapsed
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Figure 1. The enzymes in papaya latex digest the cuticle of adult Heligmosomoides polygyrus worms. (a) Latex (indicated by arrows) released from a damaged, unripe papaya. Scanning electron micrographs of the cuticular surface of adult male Heligmosomoides polygyrus after 60 mins incubation in: (b) Hanks saline in vitro; and (c) 25 mM puried papain. Note the smooth intact cuticle and prominent longitudinal cuticular ridges in (b), whereas considerable cuticular disruption is apparent with transverse ridges and sloughing of the cuticular surface in (c). Scale (b,c) 10 mm.

intervertebral discs with a similar success rate to surgery [32]. Ananain and comosain are used as debriding agents for burn injuries [33], and papain, bromelain and cin have been used as anti-inammatory drugs to replace glucocorticoids and non-steroidal anti-rheumatics [33,34]. These cysteine proteinases generally have very low oral toxicity with few or no side-effects, and an oral LD50 of . 10 g kg21 for bromelain and cin has been recorded in mice [35]. For treatment of chronic inammatory and related diseases, bromelain has been found to be safe and efcacious when administered orally in daily doses of 200 2000 mg, with the optimum dose being 750 mg per day [33,34]. Robbins [36] was the rst to report that the active anthelmintic principle of Ficus spp., named cin, was an enzyme that damaged the cuticle of Ascaris suum, presumably by proteolytic digestion. Fresh pineapple juice was found to possess an enzyme, bromelain, which is similar to cin, and which completely digested A. suum [30]. In 1940,

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Table 1. Plant cysteine proteinases and their sources

Plant species (common name) Carica papaya (papaya) Enzyme Papain Chymopapain Caricain Glycyl endopeptidase Ficin Ficain Stem bromelain Fruit bromelain Ananain Comosain Actinidain Calotropin Asclepain pH optimum 4 10 3 10 3 10 3 10 4 8.5 4 8.5 5.5 8 5.5 8 5.5 8 5.5 8 4 10 4 8 6 10 Stability to acida To pH4 To , pH1.2 To pH4 To pH3 To pH4 To pH3.3 NA NA NA NA NA NA NA Ref. [48] [49] [50] [51] [52] [36] [53] [54] [55] [56] [57] [58] [59]

Ficus carica (Mediterranean g) Ficus glabrata Ananas comosus (pineapple)

Actinidia chinensis (kiwi fruit) Calotropis gigantea (madar plant) Asclepias spp. (milkweed)
a

The ability to retain activity in digesting the cysteine enzyme-specic substrates such as benzoyl-arginyl-p-nitroanilide at pHs above the threshold indicated. Abbreviation: NA, not available.

the worm-digesting activity of a preparation of papain from C. papaya latex was described [24]. These three separate studies suggested a similar mechanism of action of rapid digestion of the Ascaris cuticle. Biochemical structure of plant cysteine proteinases The proteolytic enzymes from g, papaya, pineapple and kiwi fruit (Actinidia chinensis) (Table 1) are phylogenetically and functionally related. They all depend on a reduced thiol group of a cysteine residue as the nucleophile involved in peptide bond hydrolysis, and are thus called cysteine proteinases [37]. The cysteine proteinases of the papain family comprise a single polypeptide chain of , 25 kDa, which is folded to form a globular protein with two domains. There is a deep cleft in between the two domains where the substrates can bind, and where the active-site cysteine (Cys-25) and histidine (His-159, papain numbering) residues form a thiolate imidazolium ion pair for catalytic activity. The structures of these enzymes are very similar, and the catalytic mechanism is identical in all those studied. They can differ, however, in the amino acids lining the substrate-binding pocket, which are responsible for binding amino acid side-chains of the substrate. They could therefore differ in substrate

specicity, cleaving different peptide bonds of the substrate proteins [37] (Figure 2). Properties of cysteine proteinases All of the plant cysteine proteinases have similar, but not identical, activities, and they vary in other important characteristics such as resistance to acidic conditions and susceptibility to digestion by the enzymes of the alimentary tract (Table 1). However, specic detail is lacking in most cases and that available is focused on the limited range of sources cited. Experimental observations and clinical trials Although evidence is still lacking for other species, the cuticle of A. lumbricoides is reported to be well endowed with inhibitors of the proteolytic enzymes encountered in the alimentary tract, such as pepsin and trypsin [38], enzymes with different structures and catalytic mechanisms to the cysteine proteinases. Inhibitors that have evolved to protect the worms against proteinases of the alimentary canal have little impact on cysteine proteinase activity which, on contact, produce cuticle blistering and, eventually, total digestion of the cuticle [24,36]. Preliminary results from our laboratory corroborate this mechanism because puried papain in vitro caused blistering of the cuticle of Heligmosomoides polygyrus adult worms, rapidly followed by digestion (Figures 1b,c). This suggests, as noted by Berger and Asenjo [24], that the catalytic properties of these enzymes are fundamental to their anthelmintic effects. Some nematodes express cysteine proteinase inhibitors, although these are tissue-resident species such as Onchocerca volvulus [39] or the expression of the inhibitors is intracellular, as in H. contortus [40]. Objective in vivo assessment of these plant enzymes was neglected until the 1980s when Hansson et al. carried out a preclinical study and a clinical trial in South America to examine the effectiveness of latex from the South American g Ficus glabrata [41]. The clinical trial involved 181 residents of areas in the Amazon who were infected with one or more helminths Ascaris, Ancylostoma and/or Necator, Trichuris or Strongyloides. Upon oral administration of F. glabrata latex for three consecutive days, a reduction in the number of nematodes was achieved, although it was not evaluated statistically. No serious adverse side-effects were noted during the trial.

Figure 2. The structure of cysteine proteinases. (a) Ribbon diagram of the structure of papain. The structure is similar between the different papain family cysteine proteinases, and the catalytic-site cysteine and histidine residues (shown in the diagram) are identical. This diagram was made with the programs Molscript and Raster3d from the PDB le 9PAP [60]. (b) The solvent accessible surface of the papain molecule showing the substrate-binding cleft, running from top to bottom. The surface occupied by the active-site cysteine (yellow) and histidine (blue) residues are shown, along with the main hydrophobic-binding pocket (to bind the side-chain of a hydrophobic amino acid in the substrate polypeptide) in green. The gure was made using the Pymol [61] software package and entry 9PAP in the PDB database.
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The greatest reduction occurred with a dose of 1 ml kg21, when the worm burden was reduced by at least 50% for almost all participants infected with Ascaris or Strongyloides, by 85% for Trichuris-infected residents and by 58% for Ancylostoma and/or Necator-infected residents. There were also reductions in faecal egg counts (FEC) with a dose of 1 ml kg21: from 4381 to 815 eggs per g (EPG) for Ascaris (reduced by 81%); from 201 to 63 EPG for Strongyloides (reduced by 69%), from 673 to 371 EPG for Ancylostoma and/or Necator (reduced by 45%); and from 207 to 65 EPG for Trichuris (reduced by 69%). Mebendazole was administered to seven participants for three days as a control drug and this regime resulted in 100% reduction in Ascaris, 86% in Ancylostoma/Necator and 85% in Trichuris faecal egg counts [41]. Conrming the earlier report [36], F. glabrata latex killed Ascaris by destroying the cuticle within 24 h. Hansson et al. [41] also noted that the use of well-known, traditional anthelmintic remedies in the Amazonian region, including gs, papaya and pineapple, had decreased with the introduction of synthetic drugs. Satrija et al. [42] reported that crude papaya latex signicantly reduced faecal egg counts and the number of worms in mice infected with Heligmosomoides polygyrus, without adverse side-effects. They administered 2, 4, 6 or 8 g kg21 of crude papaya latex to mice infected with H. polygyrus and obtained a reduction in worm burden for each treatment group, which differed signicantly from the control group ( p , 0.001) and increased with increasing dose from 55.5% to 84.5% [mean worm burden in controls 76.6 ^ 5.3 (^ standard error of the mean); 34.1 ^ 4.1 in mice treated with 2 g kg21 of latex and 11.9 ^ 3.4 in mice treated with 8 g kg21 of latex]. The posttreatment faecal egg counts were lower in the treated mice when compared with untreated mice, but only in the group receiving 8 g kg21 latex was there a signicant reduction (93.3%; p , 0.01) [42]. Satrija et al. had previously administered 2, 4 or 8 g kg21 of crude papaya latex to pigs infected with A. suum. A signicant reduction in worm burden was found with the 4 g kg21 dose (80.1%; p , 0.05) and the 8 g kg21 dose (100%; p , 0.001), and also with the faecal egg count on Day 7 post-treatment (4 g kg21 dose, p , 0.05; 8 g kg21 dose, p , 0.01) [43]. These three studies [41 43] demonstrate signicant efcacy of the latex from papaya and g for treating infections with GI nematodes in monogastric hosts. New sources of plant cysteine proteinases The recognition that the anthelmintic activities in fruit extracts were probably the result of the presence of proteolytic enzymes [36] offers exciting possibilities. Out of the proteinases listed in Table 1, those from the genus Ficus have been most closely associated with anthelmintic activity. There are . 1300 known species in the genus Ficus inhabiting the tropical and subtropical regions of the world, and while not all have proteolytic enzymes in their latex [44], those that do could contain more than one proteolytic enzyme. Some of the species with high proteolytic activity grow in the tropical rainforests, the most important species being F. glabrata, F. anthelmintica and F. laurifolia [44,45]. It is noteworthy to examine other
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fruits that are related to those already known to contain cysteine proteinases. For example, kiwi fruit has not yet been investigated, and mulberry and breadfruit are both distantly related to the g. Do plant cysteine proteinases have a role as an anthelmintic? The studies, referred to herein, herald a renewed interest in the eld of herbal anthelmintics and point to a potential role for plant cysteine proteinases as acceptable anthelmintics for the treatment of GI nematode infections. However, there is a lot of work that needs to be done (Box 2). Ultimately, the only reliable means of determining relative efcacy of specic enzymes against individual species of GI nematode is an empirical one, and therefore the logical sequence is initial in vitro screening, followed by in vivo evaluation in model systems, such as H. polygyrus in mice [46]. Depending on the outcome of this phase, human clinical trials and trials in both monogastric and ruminant hosts would provide the necessary evidence for further development, applications for licence and marketing. In our view, it is unlikely that resistance to an enzyme capable of digesting the worm cuticle will arise very quickly because this would require major structural changes to the cuticle itself, or the appearance in the cuticle of a distinct proteinase inhibitor from those already present. Alternatively, cysteine proteinase inhibitors, currently only reported from internal locations in nematodes [39,40], might be diverted to secretory products and cover the cuticle, thus providing resistance to the enzymes. However, this is unlikely to be facilitated by a single genetic point mutation as in the case of resistance to synthetic anthelmintics, and hence much less likely to occur. Deciding whether any plant cysteine proteinases will prove to be sufciently effective and robust anthelmintics in the eld will depend on the outcome of the clinical trials
Box 2. Questions awaiting resolution and research priorities
Will possible seasonal variation of plants and their products prove an impediment to control, thus restricting availability of anthelmintic principles to certain times of the year? Will efcacy vary between breeds and/or strains of livestock and species of gastrointestinal nematodes? Will plant enzymes be as effective in ruminants when compared with monogastric hosts? Can in vivo efcacy be enhanced by a combination of enzyme and other treatments, or by a combination of enzymes? Can production of cysteine proteinases in the target plant species be upregulated by transgenesis or selective breeding to produce new varieties that can be grown locally for the specic purpose of worm control? Will cysteine proteinases elicit allergic reactions in some subjects or animals? Will novel cysteine proteinases from as yet undiscovered sources be more toxic than those that have been assessed and generally found to have low toxicity? How frequently will these enzymes need to be administered, and will humans and/or livestock tolerate continuous inclusion in their diets?

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and progress with addressing the questions listed in Box 2, to which currently there are no satisfactory answers. If these are encouraging, they will open up the possibility in many parts of the world for the provision of relatively inexpensive and benign anthelmintic preparations from plants growing locally. Plant-based medicines, derived from locally abundant species, are probably cheaper and more easily available to poor communities than synthetic anthelmintics, especially if plant products such as fruits, latex, leaves and stems could be grown and harvested for treatment locally. In addition, there might be fewer adverse effects when using plant-derived remedies from fruits that are natural ingredients of human and animal diets, compared with synthetic chemotherapeutic drugs [47]. The fact that cysteine proteinases are themselves proteins might also allow genetic modication of the plants to produce pharmacologically effective, or higher, doses of these anthelmintics, for direct treatment of animals and humans, or for extraction of puried enzymes.
Acknowledgements
We thank the Leverhulme Trust for a research grant held by J.M.B., D.J.B. and I.R.D. We are very grateful to John Mort, Shriners Hospital for Children, Montreal, Canada, for the structures of papain, and to Steve Ellin and Ken Cartmell, Tapton Experimental Gardens, University of Shefeld, for their help in the maintenance of g trees and in the collection of latex. We are also grateful to the anonymous referees whose constructive comments helped to improve this manuscript.

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The Malaria Vaccine Initiative four years on from Abuja

Four years ago, 43 African nations gathered in Abuja, Nigeria for a summit on malaria. Out of that summit came the Abuja Declaration, which committed the attending nations to halve the burden of malaria in Africa by 2010, and the date 25th of April was announced as Africa Malaria Day. The Malaria Vaccine Initiative (MVI; http://www.malariavaccine.org/) coordinates the efforts of a number of malaria vaccine programs with the aim of developing a cost-effective immunization strategy for the African continent. Vaccines for polio and measles save the lives of three to four million children every year, and a malaria vaccine would go a long way in achieving a large portion of the target set out at Abuja. In its effort to generate tools needed to accelerate vaccine development, the MVI is supporting a growth inhibition assay (GIA) reference centre within the National Institute of Allergy and Infectious Diseases (http://www.niaid.nih.gov/). The assay will allow the NIAID to conrm whether various blood-stage vaccines can inhibit the ability of the malaria parasite to destroy red blood cells. The MVI has also partnered with GenVec Inc. (http://www.genvec.com/) and the US Naval Medical Research Center (http://www.nmrc.navy.mil/) to assess the immunization efcacy of ve malaria antigens using a deactivated adenovirus vector. For more details, see: http://www.malariavaccine.org/040331-GENVECpress-kit.htm
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