Cheryl & ethnovet: Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura)

Volume 198, 23 February 2017, Pages 235–254

Review

http://dx.doi.org/10.1016/j.jep.2016.12.021

Abstract

Ethnopharmacological relevance

Among amphibians, 15 of the 47 species reported to be used in traditional medicines belong to the family Bufonidae, which demonstrates their potential in pharmacological and natural products research. For example, Asian and American tribes use the skin and the parotoid gland secretions of some common toads in the treatment of hemorrhages, bites and stings from venomous animals, skin and stomach disorders, as well as several types of cancers.

Overarching objective

In addition to reviewing the occurrence of chemical constituents present in the family Bufonidae, the cytotoxic and biomedical potential of the active compounds produced by different taxa are presented.

Methodology

Available information on bioactive compounds isolated from species of the family Bufonidae was obtained from ACS Publications, Google, Google Scholar, Pubmed, Sciendirect and Springer. Papers written in Chinese, English, German and Spanish were considered.

Results

Recent reports show more than 30% of amphibians are in decline and some of bufonid species are considered to be extinct. For centuries, bufonids have been used as traditional folk remedies to treat allergies, inflammation, cancer, infections and other ailments, highlighting their importance as a prolific source for novel drugs and therapies. Toxins and bioactive chemical constituents from skin and parotid gland secretions of bufonid species can be grouped in five families, the guanidine alkaloids isolated and characterized from Atelopus, the lipophilic alkaloids isolated from Melanophryniscus, the indole alkaloids and bufadienolides known to be synthesized by species of bufonids, and peptides and proteins isolated from the skin and gastrointestinal extracts of some common toads. Overall, the bioactive secretions of this family of anurans may have antimicrobial, protease inhibitor and anticancer properties, as well as being active at the neuromuscular level.

Conclusion

In this article, the traditional uses, toxicity and pharmacological potential of chemical compounds from bufonids have been summarized. In spite of being reported to be used to treat several diseases, neither extracts nor metabolites from bufonids have been tested in such illness like acne, osteoporosis, arthritis and other illnesses. However, the cytotoxicity of these metabolites needs to be evaluated on adequate animal models due to the limited conditions of in vitro assays. Novel qualitative and quantitative tools based on MS spectrometry and Nuclear Magnetic Resonance spectroscopy is now available to study the complex secretions of bufonids.

Graphical abstract

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1. Introduction

Amphibian species belonging to the family Bufonidae, most of which have parotoid macroglands behind the eyes, can be found worldwide. Commonly known as “true toads”, they are present either as native or introduced species, except in polar and extremely arid regions (Frost, 2016). In America, Asia and Europe the belly, bones, meat, skin, venom and other parts of the bufonid toads are used in the treatment of AIDS, bites, cancer, heart disorders, hemorrhages, infections, inflammation and pain (Enríquez et al., 2006, Pradhan et al., 2014, Vallejo and González, 2014 and Zhang et al., 2005). Additionally, some infections and inflammation in animals are reported to be treated with some extracts from bufonids (Gonzáles et al., 2016). These anurans can produce a sticky white secretion that is exuded not only from the parotoid glands but also from the rest of the skin (Weil and Davis, 1994) and contains a wide range of bioactive secondary metabolites of different chemotypes (e.g., Gao et al., 2010b; Grant et al., 2012; Yotsu and Tateki, 2010). Such natural products can be classified as guanidine alkaloids, lipophilic alkaloids, indole alkaloids, steroids, peptides and proteins. While guanidine alkaloids have been found only among species in the genus Atelopus Duméril and Bibron, 1841, lipophilic alkaloids have been detected in species of the genus Melanophryniscus Gallardo, 1961 ( Daly et al., 2008 and Yotsu et al., 1992). In general, the bufonid toads excrete hallucinogenic indole alkaloids; however, differences in the production patterns of these compounds have been found among genera (Sciani et al., 2013). Since the first compounds with pharmacological activity, obtained from the venom of the giant neotropical toad Rhinella marina (Linnaeus, 1758) (Bufo agua) were isolated ( Abel and Macht, 1912), more than one hundred steroidal compounds have been extracted from several species of this anuran family. Moreover, antimicrobial and anticancer peptides and proteins have been obtained from the skin and gastrointestinal tract of some Bufo Garsault, 1764 species, Duttaphrynus Frost, Grant, Faivovich, Bain, Haas, Haddad, de Sá, Channing, Wilkinson, Donnellan, Raxworthy, Campbell, Blotto, Moler, Drewes, Nussbaum, Lynch, Green, and Wheeler, 2006, and Rhinella Fitzinger, 1826, toads ( Bhattacharjee et al., 2011, Conlon et al., 1998 and Park et al., 1996).

Although only a small proportion of the more than 580 species of this family has been screened (Frost, 2016), a remarkable number of bioactive compounds has been isolated so far. This fact highlights the potential of bufonid species as an important resource for drug discovery.

2. Material and methods

2.1. Literature revision

The present article reviews the literature published prior to 2015 on toxins and bioactive natural products isolated from skins, parotoid gland secretions and other tissues of species from the family Bufonidae. Although a series of traditional remedies prepared from bufonid skin/gland secretions and crude extracts have been recorded, our pharmacological interest is focused on the isolated secondary metabolites. Therefore, only literature describing isolated chemical constituents from bufonids available in books and electronic databases such as ACS Publications, Google Scholar, Pubmed, Sciendirect and SciFinder was reviewed. For this review we only considered peer-reviewed papers with impact factor. Papers written in Chinese, English, German and Spanish were considered.

2.2. Taxonomy, nomenclature and conservation status

For the purpose of nomenclature and animal taxonomy, we consulted the websites AmphibiaWeb (AmphibiaWeb, 2016) and Amphibian Species of the World (Frost, 2016). These databases are specialized in amphibians and are frequently updated. The current taxonomy and nomenclature of each species was verified and updated accordingly using these databases, based on the scientific name and site of collection indicated in the source publication. Whenever different, the nomenclature used in the source publication is shown in parentheses.

We based the conservation status of the species included in this review on the IUCN red list categories (IUCN, 2016). According to this list, threatened categories are critically endangered, endangered and vulnerable species (IUCN, 2012). The IUCN red list categories for the 89 species considered in this review are presented in Table S-1.

3. Traditional uses of bufonids

3.1. Ethnomedicine

The use of amphibian species in the treatment of diseases by human groups from different cultures is evidence of their importance in traditional medicine. Among the 47 amphibian species reported to be used in ethnomedicinal practices, 15 species are members of the family Bufonidae (Alves et al., 2013). Remarkably, the venom of the common Asian toad species Bufo gargarizans Cantor, 1842, and Duttaphrynus melanostictus (Schneider, 1799) (Bufo melanostictus) have been used for centuries to prepare the anticancer remedy known as Chan Su and Senso in China and Japan, respectively ( Zhang et al., 2005). This medicine is offered as little brown cakes, and the quality is regulated by the Chinese Pharmacopoeia (Committee for the Pharmacopoeia of P.R. China, 2005). In addition, the empirical remedy known as Huachansu or Cinobufacini is prepared by extracting the dried skin of the toad Bufo gargarizans. This preparation which is commonly used in the treatment of various cancers, chronic hepatitis and other diseases has been approved for oral and intravenous administration according to the Chinese State Food and Drug Administration (SFDA) (ISO9002) ( Liu et al., 2014). The ethnomedicinal uses of some bufonid toads are listed in Table 1.

Table 1. Ethnomedicinal uses of Bufonidae toads in some countries.

Scientific name (Common names)	Part used	Therapeutic use	Country, Region	Preparation of the remedy	Way of administration	Reference
Bufo bufo (Rana de zarzal, Rubeta, Sapo común, Sapo negro, Zapatero)	Whole animal	Arthritis	Spain	Toad ashes mixed with rancid fat	Unespecified	Vallejo and González, 2014
	Whole animal	Cancer	Spain	Ashes of a toad (the bigest that can be found) mixed with a half ounce of ashes from verbena, romero leaves and serpentaria root	Unespecified	Vallejo and González, 2014

Bufo bufo, Epidalea calamita (Sapo negro, Zapatero)	Tadpoles	Nose hemorrhage	Spain	Ashes of tadpoles who still have tail	Unespecified	Vallejo and González, 2014
	Whole animal	Antrax	Spain, The Basque country	An alive toad has to be tied over to the pimple for eight days	Cutaneous	Vallejo and González, 2014
	Whole animal	Brucellosis	Spain, Valencia	A toad is left in the room of the patient then of two days the toad is killed and placed in the chest of the patient	Cutaneous	Vallejo and González, 2014
	Bones	Earache, toothache	Spain	A bone is pased over the area of pain	Cutaneous	Vallejo and González, 2014
	Whole animal	Tonsillitis	Spain, Galicia	Decoction of an open toad with rabbit earts	Unespecified	Vallejo and González, 2014
	Whole animal	Warts	Spain	By rubbing them with a toad	Cutaneous	Vallejo and González, 2014
	Whole animal	Herpes zoster	Spain, Almería	A live toad has to be passed nine times over the injuries. The procedure has to be repeated with two more toads.	Cutaneous	Vallejo and González, 2014
	Whole animal	Snake and spider bites	Spain	By rubbing them with a toad	Cutaneous	Vallejo and González, 2014

Bufo gargarizans (Asiatic toad, Korean toad)	Skin	Cancer, chronic hepatitis B	China	Decoction	Tablets, oral solutions and injections	Liu et al., 2014;Wang et al., 2015
Bufo gargarizans, Duttaphrynus melanostictus (Sinduria Benga)	Venom	Cancer, heart diseases, pain	China	Unespecified	Unespecified	Zhang et al., 2005
Duttaphrynus melanostictus	Skin, flesh	Gastritis	India, Western Orissa	Cooked	Orally	Pradhan et al., 2014
	Venom	Tonsillitis	India, Western Orissa	Melted	Cutaneous	Pradhan et al., 2014
	Whole body	Insect bites	India, Western Orissa	A toad is live crushed	Cutaneous	Pradhan et al., 2014

Duttaphrynus stomaticus (Dahdar)	Skin	Thelitis	Pakistan	Crushed toad skin mixed with garlic in lukewarm water	After immersion of the nipple (5 min) apply lizard oil, Saara (=Uromastyx) hardwickii	Khan et al., 2011
	Skin	Dermatitis, decubital wounds	Pakistan	Crushed toad skin mixed with garlic in lukewarm water	Apply cutaneously this solution in the affected area after washing with liquor	Khan et al., 2011
	Skin	Ripened abscess	Pakistan	Crushed toad skin mixed with garlic in lukewarm water	After drain the abscess pack it with a cotton gauze soaked with this solution	Khan et al., 2011

Incilius bocourti Brocchi, 1877 (Bufo bocourtí) (Pok’ok’, Sapo de bocourt)	Skin	AIDS	Mexico, Altos de Chiapas	Boiled	Unespecified	Enríquez et al., 2006
Rhinella jimi (Sapo cururú)	Skin, venom	Arthritis, arthrosis, asthma, backache, cancer, cough, diarrhea, earache, infections, inflammations, flu, osteoporosis, rheumatism, Sore throat, strain, toothache	Northeastern Brazil	Unespecified	Unespecified	Ferreira et al., 2012
Rhinella jimi	Venom	Cancer, gastritis	Northeastern Brazil	Unespecified	Unespecified	Alves et al., 2009
	Venom	Arthritis, bruises, inflammations	Brazil, Ceará	Unespecified	Unespecified	Ferreira et al., 2009

Rhinella marina (Giant marine toad, Sapo cururú)	Belly	Erysipelas	Brazil, Riozinho do Anfrísio	Toad alive	Cutaneous	Barros et al., 2012
	Whole body	Wounds	Brazil, Riozinho do Anfrísio	Toad macerated or toasted	Cutaneous	Barros et al., 2012

Rhinella schneideri (Sapo cururú)	Skin	Cancer	South America	Decoction	Orally	Schmeda et al., 2014
	Skin	Erysipelas	South America	Decoction	Cutaneous	Schmeda et al., 2014
	Unespecified	Acne, boils, cancer, dental caries, erysipelas, to induce abortion, urinary incontinence, wounds	Latin America	Unespecified	Unespecified	Alves R. and Alves H., 2011

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3.2. Ethnoveterinary medical uses

Toad species of the family Bufonidae have been used as a curative remedy to treat sick domestic animals in different countries (Souto et al., 2013). In particular, some bufonids have been used as medicinal remedies in horse breeding and farming of cattle. For instance, in the Brazilian region Cariri Paraibano, the tripes of the toad Rhinella schneideri (Werner, 1894) are applied topically to cure horses parasitized with Habronema muscae ( Souto et al., 2011). In Spain, the scald and hoof rot in livestock are frequently treated with products from a toad of the species Bufo bufo (Linnaeus, 1758) or Epidalea calamita (Laurenti, 1768). In the case of hoof rot, the treatment consists on cutting in half the toad and putting it directly on the infected leg. Additionally, the toad Bufo bufo is externally used to treat knee inflammation, neck troubles and wounds in horses ( Gonzáles et al., 2016). Ranchers in China and the Korean peninsula employ the meat of the toad Bufo gargarizans to treat rindertpest in the cattle species Bos indicus ( Song and Kim, 2010). The medication of injured animals with the venom from another Brazilian toad, Rhinella jimi (Stevaux, 2002), has also been reported ( Ferreira et al., 2009).

4. Guanidine alkaloids

4.1. Occurrence and source

Since the discovery of atelopidtoxin in the skin of frogs of the genus Atelopus ( Fuhrman et al., 1969), several studies have shown the occurrence of guanidine-like alkaloids in eleven species of this genus ( Daly et al., 1994 and Kim et al., 1975). Guanidine alkaloids have been found in the skin of frogs, but not in the viscera, muscle, or bone tissues (Kim et al., 1975). Moreover, this family of compounds has been found in oocytes from gravid females of A. chiriquiensis Shreve, 1936, A. glyphus Dunn, 1931, and A. zeteki Dunn, 1933 ( Pavelka et al., 1977 and Yotsu and Tateki, 2010). Although their physiological role is unknown in Atelopus frogs, guanidine alkaloids are considered to be defensive molecules against predators ( Daly et al., 1987 and Kim et al., 1975). The absence of guanidine alkaloids in other genera of the family Bufonidae, such as Bufo, Dendrophryniscus Jiménez de la Espada, 1870, and Melanophryniscus, suggests that this type of toxin is present only in the genus Atelopus ( Daly et al., 1994 and Mebs et al., 1995).

The atelopidtoxin found in the Panamanian golden frog A. zeteki, later renamed zetekitoxin, seems to consist of two toxins: the zetekitoxin-AB (1), which is more toxic than the poorly studied and not chemically defined zetekitoxin-C ( Shindelman et al., 1969, Kim et al., 1975 and Brown et al., 1977). Atelopus zeteki is the only species of the genus that secretes zetekitoxins. Nonetheless, alkaloid 1, which is an analogue of the algal toxin saxitoxin, also has been found in a population of A. varius (Lichtenstein and Martens, 1856) ( Yotsu et al., 2004). Missidentification of very similar Panamanian golden frogs, i.e., A. varius and A. zeteki, may partly explain these results ( Zippel et al., 2006). Tetrodotoxin (2) has been detected in almost all the species of Atelopus that have been tested for guanidine alkaloids (Table 2), and represents the major toxin in A. oxyrhynchus Boulenger, 1903, A. spumarius Cope, 1871, A. subornatus Werner, 1899, and A. varius ( Mebs and Schmidt, 1989, Yotsu et al., 1992, Daly et al., 1994, Mebs et al., 1995 and Yotsu and Tateki, 2010). In A. varius, the guanidine alkaloids are accountable for nearly a hundred percent of the total toxicity ( Kim et al., 1975). Another tetrodotoxin-like substance, known as chiriquitoxin (3), has been found in skin extracts from A. limosus Ibáñez, Jaramillo and Solís, 1995, A. glyphus, and A. chiriquiensis, in spite of being thought for many years to be unique to A. chiriquiensis ( Yotsu and Tateki, 2010). Guanidine alkaloids also have been found in A. ignescens (Cornalia, 1849), A. peruensis Gray and Cannatella, 1985, and A. spurrelli Boulenger, 1914 ( Daly et al., 1994 and Mebs et al., 1995). Even though the chemical compound responsible for the toxicity has not been determined in A. certus Barbour, 1923, it is thought to be a guanidine-like alkaloid ( Yotsu and Tateki, 2010). Furthermore, toxicological evaluations of skin extracts have revealed that toxins from A. cruciger (Lichtenstein and Martens, 1856) produce symtpoms similar to those from atelopidtoxin, while the effects of the skin toxins from A. planispina Jiménez de la Espada, 1875, are different from those reported for other guanidine alkaloids ( Fuhrman et al., 1969).

Table 2. Guanidine alkaloids detected in species of the genus Atelopus.

Species	Guanidine alkaloids^a					M.U. / Frog skin^b	Reference
Species	1	2	3	4	5	M.U. / Frog skin^b	Reference
A. certus						15 (9–23)	Yotsu and Tateki, 2010
A. chiriquiensis		x	x			250 (132–318)	Kim et al., 1975; Pavelka et al., 1977; Yotsu et al., 1990; Yotsu and Tateki, 2010
A. cruciger						(30–150)	Fuhrman et al., 1969
A. glyphus			x			256 (155–359)	Yotsu and Tateki, 2010
A. ignescens		x				1	Daly et al., 1994
A. limosus			x			63 (36–55)	Yotsu and Tateki, 2010
A. oxyrhynchus		x		x	x	900 (800–1000)	Mebs and Schmidt, 1989; Yotsu et al., 1992
A. peruensis		x				2	Mebs et al., 1995
A. planispina						10	Fuhrman et al., 1969
A. spumarius		x		x	x	10	Daly et al., 1994; Mebs et al., 1995
A. spurrelli		x				3	Daly et al., 1994
A. subornatus		x		x	x	50 (18–82)	Mebs et al., 1995
A. varius		x		x	x	74 (14–127)	Daly et al., 1997 and Daly et al., 1994; Kim et al., 1975; Yotsu and Tateki, 2010
A. zeteki	x	x				161 (23–418)	Brown et al., 1977; Daly et al., 1994; Kim et al., 1975; Shindelman et al., 1969; Yotsu and Tateki, 2010; Yotsu et al., 2004

a: Guanidine alkaloids in Atelopus frogs are coded as zetekitoxin-AB (1), tetrodotoxin (2), chiriquitoxin (3), 4-epi-tetrodotoxin (4) and 4,9-anhydro tetrodotoxin (5).

b: Toxicity of aqueous skin extract is shown as the average of Mouse Units (M.U.) per frog skin, followed by range in parenthesis. Values presented in TTX-equivalents were converted to M.U. (1 M U. =0.22 µg of TTX-equivalents).

Table options

The origin of tetrodotoxins in Atelopus is unclear, as they may be secreted by skin glands of the frogs, have dietary origin, or be produced by a symbiotic microorganism ( Daly et al., 1997, Mebs and Schmidt, 1989 and Yotsu and Tateki, 2010). Tetrodotoxins are known to be present in wild-caught individuals of A. subornatus and A. oxyrhynchus that lived in captivity for 3 and 3.5 years, respectively ( Yotsu et al., 1992 and Mebs et al., 1995). However, individuals of A. varius that were captive-raised from eggs did not release such toxins, suggesting that environmental factors like dietary and/or symbiotic organisms may be the natural source. This lack of tetrodotoxins in captive-raised A. varius has been considered to agree with the symbiotic hypothesis, since possible isotopically marked precursors of alkaloid 2 were not found during feeding experiments in two other species of amphibians ( Daly et al., 1997). Nonetheless, some recent evidence suggest that alkaloid 2 is produced by amphibians (Yotsu and Tateki, 2010). Because of its chemical similarity, it has been suggested that alkaloid 3 may be biosynthesized from alkaloid 2 and glycine (Yotsu et al., 1990).

The species of the genus Atelopus are known to have suffered drastic population declines and extinctions throughout Central and South America ( La Marca et al., 2005). The species mentioned in this review include one currently extinct species, i.e., A. ignescens, while the other thirteen species are considered to be threatened ( Table S-1). Consequently, further studies on these species need to be carefully conducted to avoid affecting their dwindling populations, and carried out as soon as possible, before they disappear.

4.2. Chemistry

Guanidine alkaloids in Atelopus are water soluble toxins that present some hydroxyl and guanidine groups in their structure (Fig. 1). Moreover, alkaloids 2 and 3 are better extracted with an acidic solution rather than water, suggesting that these toxins could be present as bound precursors in skin and oocytes of Atelopus ( Pavelka et al., 1977). The proton nuclear magnetic resonance (H¹NMR) spectrum of alkaloids 2 and 3, respectively, shows two diagnostic coupled doublets. In both cases, one doublet is shifted to the downfield (around 6 ppm) and the other one appears at the upfield (around 3 ppm) ( Fuhrman et al., 1976, Yotsu et al., 1990 and Yotsu and Tateki, 2010). These doublets correspond to the adjacent protons at the C-4 and C-4a positions (Fuhrman et al., 1976). Alkaloid 2 occurs at equilibrium between the hemilactal and 10,7-lactone tautomers, whereas alkaloid 3 exists mainly in the hemilactal form (Yotsu et al., 1990). The chemical structure of alkaloid 1, established by NMR correlation techniques, has been revealed to be formed by a sulfate ester, an N-hydroxycarbamate and, a 1,2-oxazolidine ring-fused lactam moiety (Yotsu et al., 2004).

: Fig. 1.
Guanidine alkaloids known from species of Atelopus.; Figure options

Guanidine alkaloids show a red color when treated with the Weber reagent, which is typical for the guanidine group (Shindelman et al., 1969). Although alkaloid 1 lacks ultraviolet absorption, this toxin can be observed as a yellow fluorescence spot at 365 nm in thin layer chromatography plates, once sprayed with an alkaline solution and heated (Shindelman et al., 1969 and Yotsu et al., 2004). Considering the small amount of guanidine toxins per frog skin, it is important to note that the mouse bioassay is 500 times more sensitive than the Weber test (Brown et al., 1977). A fluorometric HPLC method employing a post column reaction with NaOH has been applied for the identification of tetrodotoxins in some species of Atelopus ( Daly et al., 1994, Mebs et al., 1995, Yotsu and Tateki, 2010 and Yotsu et al., 1992). Once the purified toxic fractions from the frogs are obtained, electrospray ionization mass spectrometry (ESI-MS) is suitable for the identification of such toxins, where the protonated molecular ions for alkaloids 1, 2 and 3 are observed at m/z 553, 320 and 393, respectively ( Mebs et al., 1995, Yotsu and Tateki, 2010 and Yotsu et al., 2004). Additionally, when treated with tetramethylsilane under alkaline conditions, alkaloid 2 is converted into a trimethylsilyl-quinazoline derivative which is observed at m/z 407 by gas chromatography-mass spectrometry (GC-MS) ( Mebs and Schmidt, 1989).

4.3. Myotropic and neurotropic activities

A bioassay in which mice receive an intraperitoneal injection of skin extracts has determined that A. oxyrhynchus, A. glyphus, A. chiriquiensis and A. zeteki produce the most toxic extracts able to cause the death in 130 to 1000 mice per frog skin ( Table 2). The physiological effects of the alkaloids of some of these species have been studied in mammals that were treated with intravenous injections of alkaloids 1, 2, 3 and zetekitoxin-C. These four toxins are known to cause severe hypotension ( Brown et al., 1977 and Shindelman et al., 1969). Furthermore, alkaloid 1 has been observed to produce cardiac rhythm disturbances, culminating in ventricular fibrillation. Cardiac disturbance could result from effects on the sympathetic nervous system if, as suggested previously, alkaloid 1 blocks adrenergic neurons ( Brown et al., 1977 and Fuhrman et al., 1976).

Guanidine alkaloids from Atelopus act as blockers of voltage-active sodium channels (Na_v) ( Yang, 1992 and Yotsu et al., 2004). Electrophysiological studies on sodium channels of human heart, rat skeletal muscle, and rat brain expressed in Xenopus laevis oocytes showed the IC₅₀ of alkaloid 1 to be 280, 65 and 6.1 pM, respectively (Yotsu et al., 2004). This toxin apparently can act at several different sites as long as the concentration is sufficiently high (Brown et al., 1977). The potency in blocking channels by alkaloid 2 is similar to that of alkaloid 3 at pH 7.25. However, at pH 8.25, the toxin 3 is 187-fold more potent than alkaloid 2. Such an effect is thought to be caused by structural changes in the glycine residue, because both alkaloids share a similar structural base except for the glycine moiety. In addition to acting as a sodium channel blocker, alkaloid 3 also slows the activation of the potassium current in 40% of the total muscle fibers (Yang, 1992). Alkaloids 2 and 3 are known to produce a similar partial block of the action potential of a bullfrog nerve; however, alkaloid 3 decreases the conduction velocity in the nerve, while alkaloid 2 does not (Fuhrman et al., 1976). A study with several isoforms of voltage-activated sodium channels (Na_v1.2–1.8) revealed that 4,9-anhydrotetrodotoxin (5) is highly specific for the Na_v1.6 isoform, showing an IC₅₀ of 7.8 nM comparable to the potency of alkaloid 2, and suggesting that both toxins block sodium channels via the same mechanism (Rosker et al., 2007).

5. Lipophilic alkaloids

5.1. Occurrence and source

Despite being initially thought to occur only in the skin of neotropical frogs of the family Dendrobatidae, some lipophilic alkaloids have been isolated from species of the families Bufonidae, Eleutherodactylidae, Mantellidae, Myobatrachidae and Ranidae (Daly et al., 2004, Daly et al., 1984 and Rodríguez et al., 2011). These anuran species are referred to as poison frogs (Saporito et al., 2012). In the family Bufonidae, lipophilic alkaloids have been detected in skin extracts from some species of the genus Melanophryniscus. This genus contains 26 species of red-bellied toads, which are geographically distributed in Bolivia, Brazil, Uruguay, Paraguay and the northern half of Argentina ( Frost, 2016). Among the Melanophryniscus toads, the species known to be poisonous are: M. atroluteus (Miranda-Ribeiro, 1920), M. cupreuscapularis Céspedez and Alvarez, 2000, M. devincenzii Klappenbach, 1968, M. klappenbachi Prigioni and Langone, 2000, M. montevidensis (Philippi, 1902), M. moreirae (Miranda-Ribeiro, 1920), M. rubriventris (Vellard, 1947), M. simplex Caramaschi and Cruz, 2002, and M. stelzneri (Weyenbergh, 1875) ( Daly et al., 1984, Daly et al., 2007, Daly et al., 2008, Garraffo et al., 1993, Garraffo et al., 2012, Mebs et al., 2005, Mebs et al., 2007a, Grant et al., 2012, Hantak et al., 2013 and Jeckel et al., 2015). Some lipophilic alkaloids have also been identified in visceral organs, other than skin, of M. simplex at similar concentrations ( Grant et al., 2012). Among the nine toads of Melanophryniscus known to have lipophilic alkaloids, only M. devincenzii and M. montevidensis are considered to be threatened species ( Table S-1).

As in frogs of the family Dendrobatidae, feeding experiments have demonstrated that Melanophryniscus toads can sequester such alkaloidal compounds from their diet ( Daly et al., 1994 and Hantak et al., 2013). Stomach contents of some poison toads have shown a complex diet, consisting of mites and ants as the main arthropod preys ( Daly et al., 2007, Daly et al., 2008 and Quiroga et al., 2011). Some ant species from the subfamilies Myrmicinae and Formicinae contain lipophilic alkaloids ( Jones et al., 1982 and Saporito et al., 2004). Solenopsis sp., but not Formicinae ant species, were found in the stomach contents of M. klappenbachi ( Daly et al., 2008). Although Oribatidae mites are known as the source of several classes of lipophilic alkaloids, little is known about the mite taxa in the diet of Melanophryniscus species. ( Daly et al., 2007, Daly et al., 2008 and Quiroga et al., 2011).

5.2. Chemistry

More than 200 lipid-soluble alkaloids have been discovered from some toads of the genus Melanophryniscus. Such alkaloids, shown in Fig. 2, are classified as decahydroquinolines (6–12), indolizidines (13–53), piperidines (54–56), pumiliotoxins (57–88), pyrrolizidines (89–95), quinolizidines (96–101), and tricyclic alkaloids (102–103). A system of nomenclature based on their molecular weight, followed by an identifying letter which differs among alkaloids with equal molecular weight, has been used for more than 35 years for amphibian lipophilic alkaloids. Additionally, several trace lipophilic alkaloids have been detected; however, due to insufficient spectroscopy information they are referred as unclassified (Daly et al., 2005).



: Fig. 2.
Lipophilic alkaloids found in species of Melanophryniscus.; Figure options

Alkaloid fractions from poison toad extracts are frequently obtained by maceration of the skin with alcohol, followed by acid-base partition. This method generates extracts in which proteins and neutral unwanted lipids are removed. Furthermore, column chromatography and reverse-phase HPLC fractionation have been employed (Daly et al., 2008 and Garraffo et al., 1993). Low polarity columns have been extensively used for the separation and analysis of amphibian lipid-soluble alkaloids by gas chromatography/electron impact-mass spectrometry (GC/EI-MS) (Daly et al., 2007 and Mebs et al., 2005). Major fragments produced by electron impact ionization as a result of α-cleavage are observed for decahydroquinolines, indolizidines, piperidines, pyrrolizidines, and quinolizidines. Prominent ions at m/z 166, 182 and 180 are characteristic of pumiliotoxins, allo-pumiliotoxins, and homo-pumiliotoxins, respectively ( Daly et al., 1999 and Garraffo et al., 1993). Identification by gas chromatography/Fourier transformed infrared spectroscopy (GC/FTIR) has been useful in order to differentiate positional isomers, as is the case for some 3,5 di-substituted indolizidines and decahydroquinolines (Daly et al., 2005). Additionally the FTIR spectra of pumiliotoxins, allo-pumiliotoxins, and homo-pumiliotoxins show diagnostic bands at 3544, 3521 and 3555 cm⁻¹, respectively, with a shoulder at 2750 cm⁻¹ for pumiliotoxins and at 2800 cm⁻¹ for homo-pumiliotoxins (Daly et al., 1999).

Among the lipophilic alkaloids detected in Melanophryniscus species, pumiliotoxins represent the most abundant class, followed by indolizidines and pyrrolizidines ( Grant et al., 2012). Remarkably, a greater uptake efficiency of indolizidines over decahydroquinolines has been observed in M. stelzneri, suggesting some alkaloids are better sequestered from diet than others ( Hantak et al., 2013).

5.3. Myotropic and neurotropic activities

Although a great variety of lipophilic alkaloids have been identified in poison toads and frogs, few individual alkaloids have been investigated for their biological effects. The pumiliotoxin 323-A (76), a common toxin among species of Melanophryniscus, stimulates muscular and neuronal activities in mammals. This alkaloid causes marked twitches in male Sprague-Dawley rat’s phrenic nerve at 1 µM. In addition, while heart rate increment in atria strips are observed between 1.5 and 5 µM, ileum segments show rhythmic contractures and peristaltic movements at the range 0.3 – 3 µM. Both muscular tissues were obtained from male Hartley strain guinea pigs (Mensah and Daly, 1978). Moreover, alkaloid 76 stimulates influx of sodium through Na_v channels as well as altering rates of opening and closing of sodium channels in brain neurons ( Gusovsky et al., 1988 and Sheridan et al., 1991). Alkaloids 207-A (14), 209-B (15) and other 8-methyl-5-substituted indolizidines act as non-competitive blockers of nicotinic receptor channels (Daly et al., 1991). Similarly, isomers of alkaloid 223-AB (44) and some decahydroquinolines have been found to inhibit the binding of the acetylcholine-receptor antagonist [³H] perhydrohistrionicotoxin ( Warnick et al., 1982 and Aronstam et al., 1986). Remarkably, unlike most noncompetitive blockers, the potencies of the 8-methyl-5-substituted indolizidines are reduced in the presence of the agonist carbamylcholine (Daly et al., 1991).

5.4. Insecticidal activity

The pumiliotoxin 251-D (58) has insecticidal activity toward the cotton pest budworm Helliothis virescens. Although its lethal dose (0.15 µg/larvae) is 30-fold lower than that of the commercial pesticide fenvalerate, synthesis of analogues demonstrated that substituents at the side chain modulate its pesticidal potency (Bargar et al., 1995). Notably, the two 251-D enantiomers were evaluated on females of the yellow fever mosquito Aedes aegypti by measuring their abilities to feed and escape using methanol as control. The results show that the (+) enantiomer is more active than the (-) one ( Weldon et al., 2006).

5.5. Antimicrobial activity

Some of the lipophilic alkaloids, frequently found in the skin of bufonids and other amphibians, have been assayed as antibacterial and antifungal agents by employing the paper disc method. Among alkaloids, decahydroquinoline 223-F (6) was found to inhibit the growth of Bacillus subtilis and Candida albicans at 50 µg per disc. Although the effectiveness is lower than those of common antibiotics (e.g., penicillin 30 µg, tetracyclin 30 µg and nystatin 30 µg per disc), this demonstrates the potential of lipophilic alkaloids in the development of new antibiotics (Macfoy et al., 2005).

Further biological research about most of these uninvestigated lipophilic alkaloid classes is needed, because neither the mechanism of action nor toxicity and bioactivity have been studied for most of them (Daly et al., 2005). Since amphibians are in decline worldwide, known lipophilic alkaloids from amphibians have been synthetized to measure their biological effects (Toyooka et al., 2005 and Kobayashi et al., 2007).

6. Indole alkaloids

6.1. Occurrence and source

Compared with other amphibian families, the family Bufonidae represents a rich source of indole alkaloids (Cei et al., 1968 and Roseghini et al., 1986). Remarkably, one gram of parotoid gland from Incilius alvarius (Girard, 1859) (Bufo alvarius) can contain as much as 160 mg of 5-methoxy-bufotenine (104) (Erspamer et al., 1965). These alkaloids are found in the genera: Anaxyrus Tschudi, 1845, Ansonia Stoliczka, 1870, Bufo, Bufotes Rafinesque, 1815, Duttaphrynus, Epidalea Cope, 1864, Incilius Cope, 1863, Ingerophrynus Frost, Grant, Faivovich, Bain, Haas, Haddad, de Sá, Channing, Wilkinson, Donnellan, Raxworthy, Campbell, Blotto, Moler, Drewes, Nussbaum, Lynch, Green and Wheeler, 2006, Melanophryniscus, Nannophryne Günther, 1870, Rhaebo Cope, 1862, Rhinella and Sclerophrys Tschudi, 1838 ( Table S-2). With the exception of two threatened species, i.e., I. perplexus and R. atacamensis, the conservation status of species known to have indole alkaloids are regarded to be, in general, of less concern ( Table S-1). The indole alkaloid profiles among genera are different (Ceriotti et al., 1989), although species of Anaxyrus and Bufo share the same indole alkaloid pattern. For indole alkaloid profiles by species, consult the Table S-2.

Bufoviridine (105) was first reported in the green toad Bufotes viridis (Laurenti, 1768) (Bufo viridis) ( Erspamer, 1959). Species of the genus Rhaebo lack bufotenine (106), bufotenidine (107) and bufothionine (108) ( Cei et al., 1968, Mailho et al., 2014 and Sciani et al., 2013); whereas Rhinella species, besides having alkaloids 106, 107 and 108, also contain 5-hydroxy-tryptamine (109), N′-methyl-5-hydroxy-tryptamine (110), and dehydrobufotenine (111) ( Cei et al., 1968 and Maciel et al., 2006). Only alkaloid 109 has been detected in the skin and parotoid gland secretions from species of the African genus Sclerophrys. In addition to species from the genera Bufo, Bufotes, and Duttaphrynus, most of the American toads contain alkaloid 111 except those from the genera Nannophryne, Epidalea and Incilius ( Cei et al., 1968). Possible derivatives of alkaloids 104 and 106, containing sulfate attached to the indole nitrogen, have been detected in some species of Incilius ( Erspamer et al., 1967).

The most likely metabolic pathway by which the indole alkaloids are biosynthesized from alkaloid 108, in the skin of Incilius alvarius, has been proposed ( Erspamer et al., 1967). In addition, acid and alcoholic metabolites of indole alkaloids are frequently found in the skin extracts of bufonid species (Cei et al., 1968). Alkaloids 5-sulfate-bufotenine (112) and N′-methyl-5-methoxy-tryptamine (113) were detected by TLC comparisons with standards in extracts of Incilius alvarius from Arizona ( Erspamer et al., 1967).

6.2. Chemistry

Indole alkaloids found in the family Bufonidae can be conjugated at position 5 of the indole ring with a hydroxyl, methoxy or sulfate group (Fig. 3). Such basic constituents are easily extracted by polar solvents, such as methanol or ethanol. In fact, acetone is the best organic solvent for preparing a toad extract to obtain indole alkaloids (Cei et al., 1968 and Erspamer, 1959). Semi-preparative separations by employing aluminum oxide and conventional C-18 columns have been frequently obtained (Erspamer, 1959, Sciani et al., 2013 and Vigerelli et al., 2014). However, because of its indole moiety, polar copolymerized C-18 and phenyl-hexyl columns have allowed better separations (Li et al., 2014b and Zhang et al., 2005).

: Fig. 3.
Indole alkaloids found in species of the family Bufonidae.; Figure options

The ESI-MS fragmentation paterns of some of these indole alkaloids have been studied. The results revealed that a protonation in the amine nitrogen, followed by α-cleavage and hydrogen atom rearrangements, led to the formation of a common ion at m/z 160 for alkaloid 105 and its N′-methylated derivatives. While, the MS spectrum of alkaloid 104 shows a significant ion at m/z 174, thus a differentiation of 5-methoxy from 5-hydroxy substituted indole alkaloids is possible ( McClean et al., 2002).

6.3. Physiological and psychotropic activity

Indole alkaloids administered intravenously are known to cause several effects such as respiratory failure, hypertension, spleen contraction, apnea, ataxia, swaying, among other symptoms (Erspamer, 1954). The alkaloids 104 and 106 have hallucinogenic activity, the later being effective at doses of 3–5 mg when smoked (Lyttle et al., 1996 and Weil and Davis, 1994).

6.4. Muscle activity

In addition to having a relatively low diuretic capacity in hydrated rats and a potent hypertensive effect on the blood pressure of spinal cat, alkaloid 107 blocks in vivo muscular contractions induced in dog intestine by some narcotic analgesics (De Oliveira and Bretas, 1973 and Erspamer, 1952).

6.5. Anticancer activity

A recent study demonstrated that alkaloid 108 decreases the weight of liver tumors induced in male Kunming mice by modulation of the mitochondrial-mediated apoptosis response pathway, resulting in the over expression of tumor protein (p53), BH3 interacting-domain death agonist (bid), caspase-3 (Cas-3), and the down-regulation of the anti-apoptotic protein (bcl-2) in a dose-dependent manner. Additionally, it is able to improve liver histological morphology and decrease the level in blood of the liver injury marker alanine aminotransferase (ALT) in male Sprague-Dawley rats at a dose of 0.57 mg/Kg, after CCl₄ intraperitoneal administration (Xie et al., 2015).

6.6. Antiviral activity

The alkaloid 106 is able to prevent the penetration of rabies virus in the baby hamster kidney cell line, BHK-21, when tested at a concentration of 0.5–3 mg/mL. Although the mechanism of action is unclear, apparently the inhibition of the infection is through a competition for the nicotinic acetylcholine receptor (Vigerelli et al., 2014).

6.7. Antimicrobial activity

To investigate their antimicrobial potential, a variety of amphibian venom alkaloids were assayed against some bacterial and fungal pathogens. Among them, alkaloid 106 was able to produce an inhibition halo of 6, 9, 5 and 4 mm for Escherichia coli B, Proteus mirabilis, Staphylococcus aureus, and Bacillus subtilis, respectively. Its minimal effective concentration expressed as mol per mL against Proteus mirabilis is 9.2×10⁻⁶, within the average range (2×10⁻⁵ – 2×10⁻⁷) of common antibiotics such as penicillin, cephalosphorine, streptomycine and tetracycline. However, alkaloid 106 was not effective against fungi. Remarkably, the plant metabolite gremine, an analogue of alkaloid 106 without the hydroxyl group at the indole position 5, inhibits both bacterial and fungal pathogens but it lacks selectivity (Preusser et al., 1975).

7. Steroids

7.1. Occurrence and source

A great diversity of steroids have been discovered in species of Bufonidae since the initial finding of physiologically active molecules in the parotoid gland secretions from Rhinella marina and other common toads ( Abel and Macht, 1912 and Chen and Chen, 1933a). Among the steroids isolated from bufonid species are arenobufagins (114–120), artebufogenins (121–122), bufalins (123–136), φ-bufarenogins (137–139), bufatrienolides (140–145), bufogargarizins (146–147), bufotalins (148–156), γ-bufotalins (157–163), bufotricosaroids (164–165), cinobufagins (166–177), cinobufotalins (178–185), digitoxigenins (186–187), hellebrigenins (188–193), marinobufagins (194–202), norbufadienolides (203–204), norcholanes (205–209), resibufogenins (210–215), sarmentogenins (216–220), sterols (221–231), and telocinobufagins (232–244). Additionally, the steroids argentinogenin (245), bufalone (246), bufarenogin (247), bufotalinin (248), bufotalone (249), 20S, 21R-epoxy-marinobufagin (250), marinoic acid (251), marinosin (252), resibufagin (253) and 12β-hydroxy-tetrahydro-resibufogenin-3-sulfate (254) have been reported (Fig. 4).






: Fig. 4.
Steroidal compounds isolated in the family Bufonidae.; Figure options

Skin secretions of some anuran species are known to have compounds that inhibit ouabain binding to Na⁺/K⁺-dependet ATPase. The highest bioactivity levels were found among the species of the family Bufonidae (Flier et al., 1980). Chemical and biological studies of toad skin extracts revealed bufadienolides (steroids containing δ-lactone ring at carbon C-17 of ring D) as responsible agents for such activity (Lichtstein et al., 1986). Considered as possible regulators of the Na⁺/K⁺-dependet ATPase, an enzyme that controls the balance of salts and water in toad skin, bufadienolides have been detected mainly in skin and parotoid gland secretions. However, their presence in ovaries, oocytes, eggs, tadpoles, plasma and bile has been reported as well (Lichtstein et al., 1986, Lichtstein et al., 1992, Akizawa et al., 1994, Lee et al., 1994, Matsukawa et al., 1998, Mebs et al., 2007b and Hayes et al., 2009) (Table S-3). Bufadienolides are widely spread among bufonid species; however, Melanophryniscus species lack such metabolites ( Mebs et al., 2007b). An apparent exception was the report of inhibition of ouabain binding activity to Na⁺/K⁺-dependent ATPase in M. moreirae ( Flier et al., 1980 and Mebs et al., 2007b). In addition to producing bufadienolides the skin secretion of the Japanese toad Bufo japonicus Temminck and Schlegel, 1838 (Bufo vulgaris formosus), contains other steroids that alter heart rate, the so called cardenolides (steroids containing γ-lactone ring at carbon C-17 of ring D). Remarkably, this has been the only report of cardenolides and some of their conjugates with dicarboxylic acids, aminoacids, and sulfate groups in amphibians ( Fujii et al., 1975 and Shimada et al., 1977). The macrogland secretion of toads contains cholesterol (223) and lesser amounts of ergosterol (230), whose proportions differ among species (Chen and Chen, 1933a). The study by GC/MS of the sterol fraction in the venom of Duttaphrynus melanostictus allowed the identification of campesterol (222) and β-sitosterol (226) as major constituents. In addition, the steroids brassicasterol (221) and stigmasterol (227) also were detected ( Verpoorte et al., 1979). At present, more than one hundred steroids have been isolated from at least 29 species of the family Bufonidae.

Studies have demonstrated that bufadienolides in bufonid toads are biosynthesized from steroid 223 by a route other than that of plants (Porto and Gros, 1970). The bile acids bearing a 3β-hydroxy group are more efficient precursors than pregnenolone (Chen and Osuch, 1969). Although a biosynthetic pathway for bufadienolides in toads has been proposed (Porto et al., 1972), the origin of cardenolides in bufonids is unknown. However, due to the similarity in chemical structure between bufadienolides and cardenolides, an analogous metabolic pathway could be possible.

7.2. Chemistry

While cardenolides have a γ-lactone ring, bufadienolides present a δ-lactone ring which shows three diagnostic signals for protons H-21, H-22, and H-23 between 6 and 8 ppm in their ¹H NMR spectra. In both cases this moiety is found in the β orientation (Verpoorte et al., 1980). In bufonid toads, steroids can occur as free forms or bufagins and conjugates. While the free forms are relatively nonpolar compounds, the conjugates are esterified at the C-3 position with a polar moiety usually presented as a carboxylic acid, a sulfate group, or an amino acid linked to a dicarboxylic acid. In particular, the bufadienolides and cardenolides conjugated with an amino acid linked to a dicarboxilic acid, better known as bufotoxins, present arginine (Arg), glutamine (Gln), or histidine (His) as the amino acid residue.

Frequently the separation of bufadienolides by HPLC methods is carried out at 300 nm, which corresponds to the wavelength of maximum absorption by ultraviolet spectroscopy (Matsukawa et al., 1998). A rapid identification method, based on HPLC-MS, to analyze the venom constituents of the toad Rhinella marina was conducted by mixing two ionization sources in both positive and negative mode, enabling a wide mass range of identification ( Jing et al., 2013). Taking advantage of the amino acid residue ionization of bufotoxins at low pH, a positively charged C₁₈ column has been used to successfully separate bufotoxins from bufagins (Li et al., 2014a). By employing an innovative two-dimensional countercurrent-HPLC chromatography, eleven bufagins have been efficiently separated from toad venom with high purity percentages (Qiu et al., 2014). Additionally, an HPLC/MS-MS method allowed the tentative identification of nine new argininyl esters from the parotoid secretion of Rhinella schneideri, four of which were reported as natural products for the first time ( Schmeda et al., 2014). A novel experimental approach by means of acetyl choline receptor bioaffinity assessment coupled to MS analysis of skin secretion extracts from Rhinella marina and Incilius alvarius led to the identification of the bufadienolides marinobufagin (194) and marinobufagin-3-suberoyl-L-arginine ester (199) ( Heus et al., 2014).

The fragmentation pathway of bufagins by electrospray ionization in positive ion mode was studied, and certain fragmentation patterns were observed. The MS/MS spectra of bufagins can be divided into two zones: from m/z 50–250 in which appears the steroid ring fragmentation ions, and from m/z 250–600 where the fragment ions are produced by the elimination of substituent groups, such as CO and H₂O. Aditionally, positional isomers such as γ-bufotalin (157) and telocinobufagin (232) can be differentiated by comparing the relative abundance in some fragments. However, stereoisomers such as bufarenogin (247) and ψ-bufarenogin (137) could not be distinguished ( Liu et al., 2010a and Liu et al., 2010b).

7.3. Muscle activity

In etherized cats, bufagins and bufotoxins injected intravenously act as cardiotonic agents by constriction of blood vessels, followed by reduction in pulse rate, arrhythmia, tachycardia, ventricular fibrillation, and finally death. Moreover, other symptoms such as salivation, emesis and contraction of intestines and uteri are observed. (Chen and Chen, 1933a). The effect of increasing the strength of muscular contraction by bufadienolides has been related to its ability to inhibit the enzyme Na⁺/K⁺-dependent ATPase (Soliev et al., 2007). Studies on structure-activity relationship have revealed that bufagins are more potent than sulfate conjugates (Lee et al., 1994 and Shimada et al., 1987a). However, bufotoxins containing the suberoyl‐arginine group are more potent than their respective bufagins analogues (Shimada et al., 1987b and Shimada et al., 1986). In addition, the ability of marinoic acid (251) to inhibit the binding of [³H] ouabain to Na⁺/K⁺-dependent ATPase suggests that the absence of a cis C/D junction in the steroid ring is less important than the cis A/B moiety (Matsukawa et al., 1996).

7.4. Anticancer activity

In accordance with the traditional use of toad species including Bufo gargarizans, Duttaphrynus melanostictus, Rhinella jimi, Rhinella marina and Rhinella schneideri used as anticancer remedy ( Table 1); steroidal compounds from skin and parotoid gland secretions of these toads show inhibitory activity against a variety of human cancer cells (Table S-4). A cytotoxic evaluation of the steroids bufalin (123), hellebrigenin (188), 232, 226 and 250 and some chemically modified bufadienolides showed that all the steroids exhibit moderate to strong anticancer activity against the cell lines MDA-MB435 (melanoma), HCT-8 (colon cancer), HL60 (leukemia) and SF295 (glioblastoma) employing doxorubicin as the positive control. Although the steroids were cytotoxic against human peripheral blood lymphocytes, none of them caused hemolysis of swiss mouse erythrocytes. In addition, it was revealed that steroid 226 as the most active constituent against three of the four cancer cell lines tested (Cunha et al., 2010). Investigations of the anticancer potential of these toxins reveal some general structural-activity features. For example, 14β-OH steroids are more active than 14-15β epoxides and also more active than those containing the opened 17-pyrone ring. Cardenolides present weaker anticancer activity than their respective bufadienolides analogues. The lower cytotoxic value of 14α-artebufogenin (121) compared with that of its 14β-isomer suggests that a cis-A/B ring is crucial for potency (Kamano et al., 1998). In support of this, the steroids bufogargarizin-A (146) and bufogargarizin-B (147), containing an altered A/B ring, exhibit much lower anticancer bioactivity than bufadienolides (Tian et al., 2010a). Furthermore, the weak anticancer activity of bufotricosaroids and norcholanes indicates that removal of the δ-lactone ring in bufadienolides greatly decreases their antitumoral potential ( Tian et al., 2013 and Tian et al., 2010b).

The lack of hemolytic activity of bufadienolides against mouse erythrocytes, even at doses as high as 50 µg/mL, suggests that these compounds cause apoptosis without disruption of the cell membrane (Cunha et al., 2010). However, the cloroformic/methanolic (8/2) extract of the skin venom from the toad Rhaebo guttatus (Schneider, 1799) induced cell membrane disruption of human erythrocytes ( Ferreira et al., 2013). These contrasting results point out to different mechanisms for induction of cytotoxicity by bufadienolides. One via lytic activity in cell membrane, while other been carried out apoptosis. Therefore, studies on the anticancer and mechanism of action of the diferent chemotypes of this class of metabolites are nedded. The most potent of the bufadienolides, steroid 123, induces apoptosis by some signaling pathways that involve cell cycle disruption at the G₀/G₁ phase, inhibition of tube formation (angiogenesis), DNA fragmentation, down regulation of anti-apoptotic proteins and oncogenes, and activation of caspases in a way that the enzyme Na⁺/K⁺-dependent ATPase could be the initial target (Qi et al., 2011). A new apoptotic pathway has been reported by which bufagin 123 causes cell death. In the process, the ClC-3 chloride channels are activated, and the pathway PI3/ Akt/ mTOR, which is involved in cellular growth and metabolism, is downregulated (Liu et al., 2013).

7.5. Antimicrobial and antiparasitic activity

Although bufagins were formerly thought to be inactive against microorganisms, the two major components of the venom of Rhinella rubescens (Lutz, 1925) (Bufo rubescens), steroids 194 and 232, inhibit the growth of Escherichia coli (ATCC 25922) with a minimum inhibitory concentration (MIC) of 16 and 64 µg/mL, respectively. Additionally, both steroids are active against Staphylococcus aureus (ATCC 25213) with a MIC of 128 µg/mL which is even lower than that characteristic of common antibiotics, such as trimethoprim (MIC <160 µg/mL) ( Cunha et al., 2005 and Habermehl, 1995). Bioprospecting research on the parotoid gland secretions from the Brazilian toad species Rhinella jimi led to the isolation of the bugafins 188 and 232, two anti-parasitic agents with neither cytotoxicity nor hemolytic activity against mouse macrophages. While both steroids inhibit the growth of Leishmania chagasi promastigotes with IC₅₀ of 126.2 and 61.2 µg/mL, respectively, only steroid 188 was active against Trypanosoma cruzi trypomastigotes showing an IC₅₀ of 91.75 µg/mL. The ultrastructural study of L. chagasi promastigotes incubated with the bufagin 232 suggests mitochondrial damage and plasma membrane disturbances without macrophage activation as the likely mechanism of action ( Tempone et al., 2008).

7.6. Protease inhibitor activity

In addition to the bactericidal and parasiticidal activity, bufagin 232 is able to inhibit the action of the serine protease chymotrypsin at a concentration of 1 mg/mL. Although the mechanism of action was not established, it has been found that steroid 232 acts selectively since neither trypsin nor elastase were inhibited in the presence of this steroid (Shibao et al., 2015).

Owing to the well-known usage of bufonids in medicinal remedies and the proven pharmacological potential offered by the great chemical diversity of these steroids, the occurrence among species has been summarized (Table S-3). This summary table meets the need for a more complete chemical profile among species of Bufonidae (Gao et al., 2010b, Liu et al., 2014 and Zhao et al., 2014).

8. Peptides and proteins

8.1. Occurrence and source

Bioactive peptides have been isolated from the skin extracts of species from the families Alytidae, Ascaphidae, Bombinatoridae, Ceratophryidae, Dicroglossidae, Hylidae, Hyperoliidae, Leptodactylidae, Myobatrachidae, Pipidae, Ranidae and Bufonidae (Conlon et al., 2014 and Xu and Lai, 2015). Compared to other amphibians, the species of Bufonidae have been scantily bioprospected for bioactive proteins and peptides, probably because these ones occur at extremely low concentration (Rash et al., 2011). However, in addition to bioactive proteins, antimicrobial and anticancer peptides also have been isolated from skin and gastrointestinal tracts of the toads Bufo gargarizans, Duttaphrynus melanostictus, Rhinella arenarum, Rhinella marina and Rhinella schneideri.

8.2. Chemistry

Purification of bioactive extracts containing peptides and proteins is preceeded by inactivation of proteolytic enzymes with denaturing agents (Park et al., 1996). The peptidase-free homogenate is then subjected to fractionation by employing reverse phase or ionic exchange columns with ultraviolet detection (Bhattacharjee et al., 2011 and Lu et al., 2011).

Structural elucidation by Edman degradation has revealed the primary structure of some peptides and proteins from the species of bufonids. Table S-5 contains primary structure and molecular weight of peptides and proteins 255–270. Bufokinin (255), a tachykinin undecapeptide, was isolated from the toad Rhinella marina (Conlon et al., 1998). The primary structure of the neurotensin-related peptide bufo-NT (256), isolated from the same species, was determined by incubation with pyroglutamyl aminopeptidase followed by N-terminal sequencing ( Warner et al., 1998). The sequence of buforin-I (257) purified from Bufo gargarizans presents high homology to the N-terminal region of histone H2A ( Park et al., 1996). The proteins baserpin (258), bati (259), and bas-ah (260) were isolated from the toad Bufo gargarizans Cantor, 1842 (Bufo andrewsi), and their N-terminal region was sequenced. However, a more complete molecular characterization of these proteins is needed ( Zhao et al., 2005a, Zhao et al., 2005b and Zhao et al., 2005c).

In addition to primary structure sequencing, the molecular characterization also involves the determination of the molecular weight; hence, both polyacrylamide gel electrophoresis and mass spectrometry methods have been employed (Lu et al., 2011 and Sánchez et al., 2003). The complete characterization of ba-lysozyme (261), which was isolated from Bufo gargarizans, was accomplished by peptide mass fingerprinting in which the endoproteinase trypsin digests were analyzed by matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) ( Zhao et al., 2006).

8.3. Antimicrobial and antiviral activity

The antimicrobial potential of amphibians has attracted worldwide attention, and currently about 180 anuran species are known to produce antimicrobial peptides (Conlon et al., 2014 and Xu and Lai, 2015). The peptide 257 under enzymatic degradation produces Buforin-II (262). As demonstrated by comparison with the positive control, magainin 2, peptide 257 inhibits the growth of gram positive and gram negative human pathogens with MIC average values of 4.8 and 6.6 µg/mL, respectively, while peptide 262 is two-fold more potent. Furthermore, the antifungal potential of peptide 262 (1 µg/mL) is higher than that of peptide 257 (4 µg/mL) against the fungi Candida albicans, Cryptococcus neoformans and Saccharomyces cerevisiae ( Park et al., 1996). Peptide 262 penetrates the cell membrane without permeabilization by a route in which the proline residue at position 11 is crucial for its potency (Park et al., 2000). Two lectins from Rhinella arenarum (Hensel, 1867) (Bufo arenarum) named as lbp-1 (263) and lbp-2 (264) inhibit the growth of Enterococcus faecalis, Escherichia coli and Proteus morganii by forming an inhibition halo of 11–20 mm at a concentration of 25 µg/disc. The evaluation of this effect, by taking the bacteria from the inhibition halo and placing them in plates without these lectins, revealed their action occurs by a bacteriostatic mechanism (Sánchez et al., 2003). Likewise protein 261 possess antibacterial activity; however, its antimicrobial potential is higher against gram positive than gram negative organisms as juged by their MIC values determined as 1 µM and 8 µM against Staphylococcus aureus (ATCC2592) and Escherichia coli (ATCC25922), respectively. The lytic activity of protein 261, evaluated especificaly against Micrococcus lysodeikticus and determined to be 2.7×10⁵ units per mg of protein, may be like that of other lysozymes in which the enzyme disrupts the cell membrane by cleaving the β-1, 4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan (bacteriolytic) (Zhao et al., 2006). The biological role of bioactive peptides and proteins isolated from bufonid toads is still unclear. However, an analysis of the skin of Bufo gargarizans using transcriptomic techniques, allowed the identifification of a novel cathelicidin protein named bg-cath. After being cleaved, this protein generate two peptides: bg-cath-(5–37) (265) and bg-cath-37 (266). Althought these peptides present a weak potential against human bacterial pathogens, both of them are strongly active (MIC: 3.125–40 µg/mL) against the aquatic bacteria Vibrio splendidus, Streptococcus iniae and Aeromorus hydrophila, which are commonly found in the natural habitat of this toad ( Sun et al., 2015).

Although antibacterial and antifungal activity was undetectable in protein 260 even at doses up to 13 µM, this heme-containig protein inhibits HIV-1 infection and HIV-1 replication in a dose dependent maner with median effective concentration (EC₅₀) values of 0.66 and 0.83 µM, respectively, in both cases using the anti-HIV drug azidotimidina (AZT) as positive control. Taking into account its anti-HIV-1 reverse transcriptase IC₅₀ (1.32 µM) and its cytotoxic concentration values (CC₅₀: 9.5 µM) against human T cell leukemia cell line (C8166), skin secretion from bufonids represents a profilic source for antiviral medicaments (Zhao et al., 2005b).

8.4. Anticancer activity

Some proteins with antiproliferative properties have been isolated from some widely distributed and common toads. The protein bmp1 (267) isolated from the skin of Duttaphrynus melanostictus inhibits the proliferation of the leukemia cell lines U937 and K562 presenting IC₅₀ values of 49 and 30 µg/mL, respectively, as compared with the positive control, the anticancer drug 5-fluorouracil. The volume and weight of solid tumor, induced by intramusculary injection in male albino mice, were reduced in about three times after administration of protein 267 at a dose 1 mg/Kg. In addition, protein 267 was found to cause apoptosis at subG1 and G1 phases by a p53-dependent pathway, as revealed by the up-regulation of caspases 3, caspase 9 and parallel expression of bax and p53 proteins in HepG2 cells. Cytotoxicity (LD₅₀) of protein 267 was determined as 12.2 mg/Kg by intraperitoneal injection of mice ( Bhattacharjee et al., 2011 and Gomes et al., 2011).

Skin of Bufo gargarizans is the source of the anticancer peptide tsp (268). This peptide showes mild toxicity against normal liver cells (L-02) at 50 µg/mL. Moreover, a strong inhibitory activity toward liver cancer cells (HepG2) was observed when assayed in combination with 5-fluorouracil. Although cell arrest occurs at G1 phase, the mechanism of action of peptide 268 remains to be elucidated (Lu et al., 2011). Although a biological activity have been detected for proteins 267–272, their complete molecular characterization has not yet been carried out.

8.5. Muscle activity

Research on different tissues of amphibians have resulted in the isolation of peptides from different families such as neurotensins, tachykinins among others, known to have effects on muscles (Xu and Lai, 2015). The neurotensin peptide 256 is able to cause spasm in small intestine preparations of toad with an agonist potency (EC₅₀) of 8.9±0.52 nM comparable with that of the spasmogenic substance P (5.01±0.93 nM). Since this effect is not affected in the presence of the sodium channel blocker tetrodotoxin, a possible mechanism by which this peptide acts directly on the muscle cells is suspected. The contractile action of peptide 256 may involve the interaction of the C-terminal region with the muscle cell receptors as do other neurotensins (Warner et al., 1998). Tachykinin receptors, better known as neurokinin receptors (NK), are implicated in the mediation of several physiological and pathological conditions. Some neurokinin receptor antagonists have reached advanced clinical phases (Garcia and Gascón, 2015). Peptide 255 binds to the NK-1 receptor with a dissociation constant (K_d) value of 0.31±0.23 nM, showing two-fold greater affinity than the peptide substance P, which is considered to be the major NK-1 mammalian ligand. However, since a high affinity towards NK-2 (K_d: 2.79±0.46 nM) and NK-3 (K_d: 47.6±13.7 nM) receptors was also observed, peptide 255 lacks selectivity. Structure-activity relationship studies have revealed that a proline residue at position 4 and an aspartate residue at position 5 are important for high binding affinity for NK-1 and NK-2 receptors, respectively (Conlon et al., 1998).

8.6. Immunomodulatory activity

The innate defensive machinery better known as the complement system plays important role in host defense and inflammatory processes. Defficiencies in either activation or inactivation of this protein system can lead to several diseases such as Alzheimer’s, asthma, lupus, sclerosis, and other affections (Sarma and Ward, 2011). In this regard, the proteins s-2 (269) and s-5 (270), isolated from Rhinella schneideri, have been found to active the complement cascade by stimulating the generation of C3, C3a, and C5a fragments in normal human serum, according to the inmunoelectrophoresis and neutrophil chemotaxis assays. Furthermore, protein 269 was observed to induce the production of the terminal complement complex SC5b-9 by the ELISA technique. The dose of action for each protein was determined as the absorbance at 280 nm contained in 50 µL, thereby the dose of protein 269 is 0.2 and that of protein 270 is 0.16 (Anjolette et al., 2015).

8.7. Protease inhibitor activity

The proteins 258 and 259 from Bufo gargarizans, two single chain glycoproteins, are able to inhibit proteases. While protein 259 acts reversibly against trypsin with an inhibition constant of 14 nM, protein 258 forms an SDS-PAGE-stable covalent complex when inhibits bovine trypsine, bovine chymotrypsin and porcine elastase having association constants of 4.6×10⁶ M⁻¹ s⁻¹, 8.9×10⁶ M⁻¹ s⁻¹ and 6.8×10⁶ M⁻¹ s⁻¹, respectively. The N-terminal sequence of both proteins shows no similarity with previously known protease inhibitors. Since no antimicrobial activity was detected, these proteins are thought to act as inhibitors against certain endogenous proteases of this toad ( Zhao et al., 2005a and Zhao et al., 2005c).

9. Other compounds

In addition to the above cited compounds, other bioactive constituents that do not fit into these aforementioned categories have also been detected in skin extracts of some bufonid species (Fig. 5).

: Fig. 5.
Other bioactive compounds isolated from Bufonidae species.; Figure options

9.1. Anti-inflamatory activity

The phenolic compound hydroquinone (271), which is the active principle of the venom from the beetle Palembus ocularis, was detected by GC/MS in alcoholic skin extracts of a few species of Melanophryniscus toads from the northern and south-eastern area of Uruguay. Analysis of eggs and tadpoles of these poison toads revealed no occurrence of phenol 271, pointing to a dietary origin ( Mebs et al., 2005 and Mebs et al., 2007a). Biological assays showed that phenol 271 shows anti-inflammatory activity by inhibiting the 5-lipoxygenase enzyme (Wahrendorf and Wink, 2006).

9.2. Analgesic activity

The skin and other tissues of some vertebrates have been screened for nonpeptide opioids. The skin of the toad Rhinella marina produces large quantities of a nonpeptide opioid compound (3.01±1.48 pmol/g of toad skin). The isolation, guided by immunological and chromatographic criteria, in addition to the analysis by GC/MS, allowed the identification of morphine (272) ( Oka et al., 1985).

9.3. Sleep inducing activity

A compound isolated from the methanol skin extract of Duttaphrynus melanostictus, that induces sleep in male albino mice, was named as sleep inducing factor (SIF). An intraperitoneal administration of the compound SIF at 4 mg/Kg was found to increase in 7 times the amount of sleep. In addition, SIF failed to cause lethality by intravenuous administration at a dose of 10 mg/Kg. The molecular weight of SIF was determined to be 880 Da. Although its chemical structure is still unknown, spectroscopy data suggest SFI is a conjugated aromatic compound with carbonyl and hydroxyl functional groups (Das et al., 2000).

10. Toxicity

Pharmacological evaluations in mammals revealed the toxic effects of chemical constituents of secrections from bufonid species. In the case of steroids, an aqueous-alcoholic dilution, injected in etherized cats, caused the death with an average fatal dose of 0.19 and 0.40 mg/Kg for bufagins and bufotoxins, respectively (Chen and Chen, 1933a). The cytotoxicity concentration (CC₅₀) of the protein 260 was evaluated on human leukemia cells (C8166) and determined to be 9.5 µM by the MTT colorimetric method (Zhao et al., 2005). A great diversity of lipophilic alkaloids have been identified among Melanophryniscus toads; however, their toxicity is unknown. While, the guanidine alkaloids 1–3 show citototoxicity against mice. Their LD₅₀ values, obtained by intravenous injection are 11, 10 and 14 µg/Kg, respectively ( Brown et al., 1977 and Yotsu and Tateki, 2010). Studies with human volunteers being administrated with the indole alkaloid 106 as bufotenine-oxalate showed no visual effects, neither at 4 mg applied intravenuously nor up to 16 mg applied intranasally. However, anxiety and perceptual changes were reported at 8 mg by intravenuous injection (Lyttle et al., 1996). In study carried out with mice, these died after intraperitoneal injection of 0.5 mg of the alkaloid 106, possibly due to respiratory failure (Erspamer, 1954).

To our knowledge, there is no confirmed report of poisoning in humans during traditional treatments by employing medicine prepared from bufonid species. Nonetheless, the death of a 40 year old man who consumed aphrodisiac pills, suspected to be based on toad venom, was reported. The patient showed sore throat, emesis, pain in the gastric region and first degree ventricular block. In addition the metabolic profile revealed a high serum potassium concentration (7.6 mM), which is a typical feature in toad poisoning (Gowda et al., 2003).

In spite of the reported cardiotoxicity of bufonid secretions, a toad skin-based remedy used in Chinese traditional medicine “Huachansu” is used in the treatment of cancer. This preparation is given at doses of 20–25 mL or 20 mL/m². A pilot study on eleven patients with advanced cancer was conducted by injecting Huachansu at 10, 20, 40, 60, and 90 mL/m² intravenously. The concentration of the reported main constituents of Huachansu in the injection was as follows: steroid 123 (14.3±0.03 ng/mL), cinobufagin (166) (3.35±0.1 ng/mL), cinobufotalin (178) (21.5±0.22 ng/mL), and resibufogenin (210) (24.5±2.18 ng/mL). As result, no toxicities were found during the first cycle of the therapy and 40% of the patients had stable disease. Remarkably, one individual with hepatocellular carcinoma which has received 8 cycles at a dose of 10 mL/m² presented 20% reduction in tumor mass (Meng, et al., 2009). Despite there are not many studies involving compounds from bufonid frogs in humans the examples described above for compounds 106, 123, 166, 178, 210 suggest and acceptable risk-benefit ratio for such studies.

11. Clinical trials

To our knowledge there are not reports on compounds from bufonids in clinical trials.

12. Concluding remarks

Research on the use of bufonids for the treatment of several diseases has been based in their ethnomedicinal use, and in vitro investigations for inflammation and cancer. The evaluation of compounds in vivo that leads to a deeper understanding of their pharmacology and potential use as drugs for the treatment of human and animal diseases; is a significant challenge of this field, which needs to be addressed. However, the results obtained so far point out that bufonids posess a great potential to search for new compounds with a wide range of biomedical applications. An urgency for research and development of novel therapeutics based on anurans comes from the fact that more than 30% of amphibian species are in decline, and about 160 species are considered to be extinct (IUCN, 2016). Nowadays it is possible to study their secondary metabolites using noninvasive techniques such as chemical, electrical or mechanical stimulation (Clark, 2010). These allow the collection of secretions and the subsequent release of the species back into their natural habitat. Thus, it may be possible to better characterize and understand these potentially valuable therapeutic compounds including guanidine, indolic and lipophilic alkaloids, steroids, peptides and proteins without contributing to amphibian declines. Taken together, these remarks highlight the importance of studying amphibian-derived compounds with care for the conservation of amphibians and their diversity. Nonetheless, we may be losing many potentially valuable compounds and knowledge as these amphibian declines and extinction advances.

Acknowledgements

We gratefully acknowledge the National Secretariat for Science and Technology of Panama (SENACYT) throught the INDICASAT-BID program (grant numbers IND-JAL-05 and 02-12-H). RI, MG and AD were supported by the National System of Research (SNI). In addition, RI was supported by the Panama Amphibian Rescue and Conservation Project, and CR by a joint scholarship from Instituto para la Formación y Aprovechamiento de Recursos Humanos (IFARHU) and SENACYT. LR-S was supported by the US National Science Foundation grant IOS-1121758.

Appendix A. Supplementary material

Supplementary material. Table S-1: Conservation status of the reviewed species.

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Supplementary material. Table S-2: Indole alkaloids detected in toads of the family Bufonidae.

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Supplementary material. Table S-3: Steroids isolated in toads of the family Bufonidae.

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Supplementary material. Table S-4: Anticancer activity of steroids isolated from Bufonidae species.

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Supplementary material. Table S-5: Peptides and proteins from toads of the family Bufonidae.

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Tuesday 31 January 2017

Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura)

Abstract

Ethnopharmacological relevance

Overarching objective

Methodology

Results

Conclusion

Graphical abstract

1. Introduction

2. Material and methods

2.1. Literature revision

2.2. Taxonomy, nomenclature and conservation status

3. Traditional uses of bufonids

3.1. Ethnomedicine

3.2. Ethnoveterinary medical uses

4. Guanidine alkaloids

4.1. Occurrence and source

4.2. Chemistry

4.3. Myotropic and neurotropic activities

5. Lipophilic alkaloids

5.1. Occurrence and source

5.2. Chemistry

5.3. Myotropic and neurotropic activities

5.4. Insecticidal activity

5.5. Antimicrobial activity

6. Indole alkaloids

6.1. Occurrence and source

6.2. Chemistry

6.3. Physiological and psychotropic activity

6.4. Muscle activity

6.5. Anticancer activity

6.6. Antiviral activity

6.7. Antimicrobial activity

7. Steroids

7.1. Occurrence and source

7.2. Chemistry

7.3. Muscle activity

7.4. Anticancer activity

7.5. Antimicrobial and antiparasitic activity

7.6. Protease inhibitor activity

8. Peptides and proteins

8.1. Occurrence and source

8.2. Chemistry

8.3. Antimicrobial and antiviral activity

8.4. Anticancer activity

8.5. Muscle activity

8.6. Immunomodulatory activity

8.7. Protease inhibitor activity

9. Other compounds

9.1. Anti-inflamatory activity

9.2. Analgesic activity

9.3. Sleep inducing activity

10. Toxicity

11. Clinical trials

12. Concluding remarks

Acknowledgements

Appendix A. Supplementary material

References