Recent Developments in the Field of Arrow and Dart Poisons

Home , Arrow poison, Cardiotoxicity, Choresine, Phyllobates aurotaenia, Phyllobates bicolor

Journal of Ethnopharmacology 100 (2005) 85–91

Perspective paper Recent developments in the ﬁeld of arrow and dart poisons Genevieve` Philippe, Luc Angenot ∗

University of Liège, Natural and Synthetic Drugs Research Centre, Laboratory of Pharmacognosy, B36, Avenue de l’Hôpital 1, 4000 Liège, Belgium Accepted 18 May 2005 Available online 1 July 2005

Abstract

Arrow and dart poisons, considered as conventional natural sources for future drug discovery, have already provided numerous biologically active molecules used as drugs in therapeutic applications or in pharmacological research. Plants containing alkaloids or cardiotonic glycosides have generally been the main ingredients responsible for the efficacy of these poisons, although some animals, such as frogs, have also been employed. This paper, without being exhaustive, reports the greater strides made during the past 15 years in the understanding of the chemical nature and biological properties of arrow and dart poison constituents. Examples both of promising biological properties shown by these molecules and of crucial discoveries achieved by their use as pharmacological tools are given. Further studies of these toxic principles are likely to enable scientists to find new valuable lead compounds, useful in many fields of research, like oncology, inflammation and infectious diseases. © 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Dart poisons; Arrow poisons; Alkaloids; Cardiac glycosides; Frog skin

1. Introduction Even if monovalent poisons are described, more often they consist of a mixture of materials. Plant extracts con- During the course of their evolution, many plant species taining alkaloids, diterpenoids or triterpenoids, including have survived through their ability to synthesize and accu- especially the cardiotonic glycosides, are generally the main mulate toxic compounds that protect them against microor- toxic compounds responsible for the efficacy of arrow and ganisms, insects, herbivores or even other plants. Some of dart poisons. However, a large number of adjuvants are these toxins have found a role in the subsistence activities also added for a variety of reasons: to thicken the extract of traditional societies, providing a source of arrow and fish and enable it to adhere better to the arrow, to augment poisons. In nearly all parts of the world, hunting poisons pre- the effectiveness of the poison, for magical purpose or pared from extracts of poisonous plants have been used as to hide the real composition of the poison. Besides the auxiliary weapons in the procurement of food and clothes. use of plant extracts, a few ingredients are obtained from Indeed, various peoples made their hunting trips more effec- animals. tive by applying poisonous materials to their arrows, darts, In subsistence communities, some of these poisons have spears or javelins. These poisons have also been employed also been employed as ingredients in or sources of traditional in preventing the depredations of wild animals, or for martial remedies. During the last century, the chemical and pharma- purpose. Frequently, the same poisons were used in ordeals. cological study of their active principles led to the discovery This involved the administering of a plant poison to the sus- of famous medicines still used in western countries, the most pect and the subsequent determining of guilt or innocence convincing example of which being the different types of depending on the poison’s effects (e.g. vomiting, urination, curare (Bisset, 1992a). Arrow and ordeal poisons are still con- ... ). Such products continue to be utilized locally, although sidered today as conventional natural sources for future drug to an extent difficult to determine. discovery (Tulp and Bohlin, 2004). Moreover, they have been and still are an important source of pharmacological tools, as ∗ Corresponding author. Tel.: +32 4 366 4330; fax: +32 4 366 4332. well as a valuable source of original chemical structures for E-mail address: [email protected] (L. Angenot). the development of new drugs.

In his excellent review written in 1989, Bisset reported different types of toxic mechanisms are associated with the the history of arrow and dart poisons and the geographical Strychnos species: a tetanising activity caused by strychnine distribution of their use. He also considered their chemical (1) and some of its monomeric derivatives and a paralysing composition, the pharmacological properties of their active action due to a series of quaternary alkaloids included in principles and their applications as medicinal agents or phar- curare poisons. They are also among the most renowned macological tools (Bisset, 1989). In this paper, we would plants of traditional pharmacopeias. like to mention the main discoveries in the ﬁeld of arrow and During the last few decades, different pharmacologi- dart poisons that have occurred since the publication of this cal properties have been described for Strychnos alkaloids outstanding document. (Fig. 1) or extracts (Philippe et al., 2004). For example, recent in vitro screenings of Strychnos species for antiplasmodial activity have led, after bioguided puriﬁcations, to 2. Plant sources the isolation of new bisindole alkaloids with promising in vitro activities against chloroquino-resistant strains of Plas- Among the different plant genera listed as ingredients of modium falciparum (Fred´ erich´ et al., 2002). In particular, dart and arrow poisons, the most active are known to contain antiplasmodial sungucine (2)-type alkaloids have been iso- biodynamically active alkaloids or cardiotonic glycosides, lated from the distinctively red-coloured roots of Strychnos and it seems that members of the other genera function mostly icaja Baillon, widely used in ordeal and arrow poisons in as additives. The latter will not be discussed here. West and Central Africa and traditionally employed to treat malaria. The most active alkaloid of this type to date has been 2.1. Alkaloids strychnogucine B (3)(Fred´ erich´ et al., 2001). Usambarine- type alkaloids, such as isostrychnopentamine (4), have also Because of their toxicity, many Strychnos species have been isolated from Strychnos usambarensis Gilg, the main been used as arrow poisons or in ordeals. Two completely ingredient of an African curarising arrow poison. The in

Fig. 1. Structures of alkaloids from Strychnos species: strychnine; sungucine and its most antiplasmodial derivative, strychnogucine B; strychnohexamine; isostrychnopentamine; guiaflavine. G. Philippe, L. Angenot / Journal of Ethnopharmacology 100 (2005) 85–91 87 vitro and in vivo antimalarial properties of isostrychnopen- was also the first report on Clathrotropis species being used tamine (4) have been demonstrated (Fred´ erich´ et al., 2004). for preparation of arrow poison (Sagen et al., 2002). Its proapoptotic properties have also been shown at a concentration 50–100 times higher than its antiplasmodial properties 2.2. Cardiotonic glycosides (Fred´ erich´ et al., 2003). In addition, some years ago, the first naturally occurring “trimeric” indolomonoterpenic alkaloid, Even though their therapeutic properties have remained called strychnohexamine (5), possessing mild antimalarial unknown for a long time, the cardiotoxicity of numerous properties, was also isolated from Strychnos icaja (Philippe plants containing cardiotonic glycosides has been used in et al., 2002). arrow poisons from the earliest times. In patients with heart Surprisingly, the antinociceptive effects of crude alka- failure, cardiac glycoside treatment improves symptoms and loid fractions of processed and unprocessed strychnine- reduces the need for hospitalization without affecting mortal- containing Strychnos nux vomica L. seeds in different anal- ity. In 1997, Jens Christian Skou was awarded the Nobel Prize gesic tests in mice have been studied: the extracts seemed in chemistry for his discovery of the Na, K-adenosine triphos- to be about 1000 times more potent than morphine (Cai et phatase (Na, K-ATPase or Na, K-pump), which is specifically al., 1996). Strychnine (1), one of the most famous poisons, is inhibited by cardiac glycosides. While there is no doubt that now a frequently used tool in neuropharmacological science cardiac glycosides are safe and effective in heart failure, the (Rajendra et al., 1997): this alkaloid labels specifically the underlying mechanisms are still debated (Schwinger et al., glycine receptor, which is often called “strychnine-sensitive 2003). glycine receptor”, to avoid confusion with other receptors Besides their cardiotonic properties, the biological prop- that glycine modulates. erties of cardenolides, and not only those of digitalis (not Many scientists have been fascinated by curare (Bisset, used in arrow poisons), could be very useful in other medi- 1992b). Recently, Strychnos guianensis (Aubl.) Mart., the cal fields, such as in the treatment of inflammation or cancer. first botanically identified plant of South American curares, Ouabain (7), the rapidly active glycoside found in African and one of the most common ingredients of curares, has Strophanthus-based poisons, is frequently used in research been reinvestigated. This work has led to the isolation of sev- (Fig. 2). Its use is not only as a cardiotonic drug, but also as eral bisindole quaternary alkaloids, guiachrysine, guiaflavine a pharmacological tool, especially in the study of the role of (6) and their 5-6-dehydrogenated derivatives (Penelle Na/K-ATPase (which seems to be involved in many biologi- et al., 2001). These asymmetrical alkaloids were previously cal processes) or the role of endogenous cardiotonic steroids unidentifiable. Indeed, their structure determination was (ECS), the putative ligands of the inhibitory binding site of achieved by recent advances in NMR techniques (Phillipson, this membrane sodium pump (Bereta et al., 1995; Xie, 2001; 1995). In vitro, these alkaloids, and especially guiachrysine, Dmitrieva and Doris, 2002, 2003; Masocha et al., 2003; were shown to be very effective antagonists of the human Akimova et al., 2005). nicotinic acetylcholine receptor (n-AChR), only 2–5 times Cardiac glycosides are now considered as potential anti- less potent than d-tubocurarine, with a similar mechanism of cancer drugs. It has been shown that digitoxin in clinically action (Wins et al., 2003). relevant concentrations induces apoptosis in different human Recently, quinolizidine alkaloids have been found in malignant cell lines in vitro. This apoptosis-inducing capa- the bark of Clathrotropis glaucophylla Cowan, of the bility seems to be explained by complex dose-dependent Fabaceae family, used as admixture of curare arrow poison mechanisms of action involving many signalling systems, in Venezuela. This was the first time quinolizidine alkaloids and not only Na, K-ATPase inhibition (Haux, 1999, 2002). have been isolated from an arrow poison ingredient and it In a digitoxin user population, there is a relationship between

Fig. 2. Structures of ouabain and 2-oxovoruscharin, two cardiotonic glycosides. 88 G. Philippe, L. Angenot / Journal of Ethnopharmacology 100 (2005) 85–91 a high plasma concentration of digitoxin and a lower risk semisynthetically derived from 2-oxovoruscharin (8) dis- of leukaemia/lymphoma and of cancer of the kidney/urinary play potent in vitro anti-tumour activity and a high level tract (Haux et al., 2001). The potential value of cardiac gly- of in vivo tolerance (van Quaquebeke et al., 2005). The cosides for the treatment of some types of human cancer is anti-inflammatory compounds of the latex seem to be of a supported by recent studies: for example, interest in ouabain proteinaceous nature (Alencar et al., 2004). Using mice mod- (7) is demonstrated in androgen-independent prostate cancer els, a recent study has concluded that the protein fraction (Huang et al., 2004) and interest in ouabain (7), digoxin and derived from the whole latex of Calotropis procera possesses digitoxin is shown in the area of breast cancer (Kometiani antinociceptive activity, which is independent of the opioid et al., 2005). system (Soares et al., 2005). The targeting by cardiac glycosides of different pro- The highly toxic latex from Antiaris toxicaria (Pers.) inflammatory mediators also represents an attractive Lesch., belonging to the plant family Moraceae, is well approach for the development of new therapeutic strategies known as one of the major ingredients of arrow and dart to control inflammation. It has been observed that ouabain, poisons made in different parts of Asia. Its toxicity is due when bound to Na/K-ATPase, converts the enzyme into a to a complex mixture of cardenolide glycosides, which con- signal transducer and initiates multiple gene regulatory path- tinues to be investigated. An investigation of nine Malaysian ways (Xie, 2001). Although ouabain (7) induces activation of dart poisons has confirmed that their main active compo- the nuclear transcriptor factor-kappa (NF-kappa B) and stim- nents are cardenolides from Antiaris toxicaria and alkaloids ulates VCAM-1 and iNOS expression (Bereta et al., 1995), probably from different forms of Strychnos ignatii Berg. Two other cardiotonic glycosides seem promising for the treat- new cardiac glycosides have been isolated and their structures ment of inflammatory diseases. By suppressing activation determined (Kopp et al., 1991). Recent bioassay-guided frac- of NF-kappa B, oleandrin can suppress inflammation and tionation of the extract of a dart poison from Indonesian Bor- perhaps tumorigenesis (Manna et al., 2000). Digitoxin also neo, derived from Antiaris toxicaria, has led to the isolation of deserves consideration as a candidate drug for the treatment three new cardenolides, toxicarioside A (Carter et al., 1997a), of inflammatory diseases, such as cystic fibrosis (because and its isomers, toxicarioside B and toxicarioside C (Carter of its ability to suppress IL-8-dependent lung inflammation) et al., 1997b). Other less studied genera of the same botan- (Srivastava et al., 2004). ical family were also investigated a few years ago, which Once again, chemists have been inspired by nature and are allowed the structural determination of two new glycosides now trying to synthesize modified cardenolides with better (Shrestha et al., 1992). The latex from Maquira Aublet and therapeutic indices or with specific properties (for instance, Naucleopsis Miq. species has been used in South America inhibition of human cytochrome P450 (17alpha)) (Schneider (in Colombia, Ecuador and north-west Brazil) as a source and Wolfling, 2004). Moreover, considering that cardiac gly- of dart poison. The principal cardiac glycosides present in cosides are often present in arrow and dart poisons and that Maquira species are strophanthidin-based and the main ones numerous plant species involved in arrow poisons have not occurring in Naucleopsis species are antiarigenin- as well as yet been investigated or only partially so, many molecules strophanthidin-based. with probably original structures could still be isolated from these hunting poisons. Most of the cardiac glycoside arrow-poison plants (Stro- 3. Animal poisons phanthus, Acokanthera, ...) belong to the Apocynaceae but other families are also important sources of cardenolide- Animals (such as arthropoda, amphibians, snakes, ...) based poisons (Bisset, 1989; Neuwinger, 1996). Calotropis have been the source of extremely potent toxins used in dart procera (Ait.) R.Br., a member of the Asclepiadaceae, is a and arrow poisons. These generally act at single or multi- laticiferous shrub. In African countries, its latex is used in ple sites of the neuromuscular apparatus (Senanayake and arrow poison. Calotropis procera is also a herbal drug used Roman, 1992). Their study has led to the isolation and deter- in Ayurvedic medicine. The cytotoxicity of extracts of its mination of valuable lead candidates for novel drugs. The root, leaves and flowers has been shown (Smit et al., 1995), specific actions of these toxins are being widely exploited as well as the anti-inflammatory activity of its latex (Kumar in the study of neuromuscular physiology and pathology. In and Basu, 1994). Different teams of scientists have identified some countries, arrowheads have been dipped in decompos- numerous cardenolides, including uscharin and voruscharin, ing animal or human flesh but, in those cases, the toxicity is first isolated by Hesse and collaborators in 1939 and 1957 rather microbial in origin and will not be discussed here. respectively (Bruschweiler¨ et al., 1969; Cheung et al., 1983), In South America, three frogs of the family Dendro- and 2-oxovoruscharin (Fig. 2)(8)(van Quaquebeke et al., batidae, Phyllobates bicolor, Phyllobates aurotaenia and 2005). Among these, two are protected by patents: uscharin Phyllobates terribilis, secrete toxins ranking among the (Anand et al., 2002) and 2-oxovoruscharin (8) (and ana- most powerful animal poisons known: batrachotoxin (9) logues thereof) (van Quaquebeke et al., 2004). Briefly, both and homobatrachotoxin (Fig. 3). These steroidal alkaloids, the inventions relate to the use of these molecules as medica- already mentioned by Bisset (Bisset, 1989), are potent ments for treating cancer. In particular, novel cardenolides modulators of voltage-gated sodium channels, leading to G. Philippe, L. Angenot / Journal of Ethnopharmacology 100 (2005) 85–91 89

Fig. 3. Structures of poisonous frog alkaloids, batrachotoxin and epibatidine. irreversible depolarisation of nerves and muscles, fibrilla- of pumiliotoxins has been demonstrated in two genera of tion, arrythmias and eventually cardiac failure. In particular, formicine ants, Brachymyrmex and Paratrechina, as well batrachotoxin (9) profoundly modulates the voltage-gated as the presence of these ants in the stomach contents of sodium channel Nav1.8, which is considered to be a key the pumiliotoxin-containing dendrobatid frog, Dendrobates player in nociception (Bosmans et al., 2004). pumilio (Smith and Jones, 2004). Evidence now indicates The isolation of batrachotoxin (9) and its derivatives has that poison frogs have an efficient system that accumulates opened the way for the study of poisonous frogs as sources alkaloids from dietary alkaloid-containing arthropods. This of novel useful chemical structures. Hundreds of alkaloids explains the fact that frogs have been found to completely lack have been identified in extracts from frog skins, including skin alkaloids when raised in captivity (Daly et al., 1994). batrachotoxins, pumiliotoxins (displaying myotonic and cardiotonic activities), histrionicotoxins (non competitive block- ers of nicotinic receptor channels and potassium channels), 4. Conclusion gephyrotoxin (blocking nicotinic acetylcholine receptor-ion channel complex) and epibatidine (Fig. 3)(10)(Daly, 1998; Indigenous tribes all over the world have discovered Patocka et al., 2000). One of the greatest discoveries over numerous plants and also animals with highly toxic prop- the past 15 years has been the isolation of epibatidine from erties, useful for hunting and protection from wild animals the skin of Ecuadorian poison frog, Epipedobates tricolor or enemies. The study of these arrow and dart poisons has (Spande et al., 1992). An increasing number of studies are led to the discovery of valuable drugs still used today (such devoted to the powerful non-opioid analgesic properties of as curare), because toxicity generally reflects high biolog- this extremely potent and selective nicotinic agonist (Qian et ical activity, the secret of an important remedy resting in al., 1993; Gerzanich et al., 1995), which have led to major the applied dose! Arrow and dart poisons nowadays pro- vide chemists and pharmacologists with original chemical interest in the use of ␣7 agonists in pain (Li et al., 2005). Epi- batidine (10) is the most powerful natural nicotinic agonist structures available for pharmacological screening and/or known. Different ways of total synthesis have been described for semisynthesis. Many are important drugs or tools in and interestingly, both the (+)- and (−)-isomers of synthetic hot fields of research, like oncology, inflammation, neuro- epibatidine possess similar biological activity. This alkaloid science or infectious diseases. Chemical substances derived of a new class has become an established source of new from natural products have always been a major source of drugs and has served as the lead in the design of new lig- lead compounds for the pharmaceutical industry (Newman ands selective for distinct nicotinic receptor subtypes (for et al., 2003). Furthermore, crucial breakthroughs in sepa- example: Wei et al., 2004; Baraznenok et al., 2005). More- ration and structure-determination technologies (Koehn and over, this alkaloid shows high binding affinity to nicotinic Carter, 2005) are now leading more easily to the isolation acetylcholine receptors and is, when radioactively labelled, and identification of complex molecules that the chemist is an important neuropharmacological tool for the study of the not always able to synthesize. Nevertheless, it is important distribution and characterization of nicotinic receptors (Gotti that ethnopharmacologists survey indigenous tribes widely and Clementi, 2004). before they completely lose their traditional culture. We hope Batrachotoxin alkaloids have also been found in passer- that these examples of promising natural molecules empha- ine bird species endemic to New Guinea (Dumbacher size how it is essential, for the provision of future effective et al., 1992). The presence of high levels of batrachotox- drugs, to preserve the ecological systems. ins in a group of beetles, genus Choresine (family Melyri- dae), found in the stomach contents of the birds suggests that they sequester these alkaloids from dietary sources. The References cosmopolitan family, Melyridae, could be the source of the Akimova, O.A., Bagrov, A.Y., Lopina, O.D., Kamernitsky, A.V., Trem- batrachotoxins found in the highly toxic Phyllobates frogs blay, J., Hamet, P., Orlov, S.N., 2005. Cardiotonic steroids differen- (Dumbacher et al., 2004). In the same way, the presence tially affect intracellular Na+ and [Na+](i)/[K+](i)-independent sig- 90 G. Philippe, L. Angenot / Journal of Ethnopharmacology 100 (2005) 85–91

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