**5. Biotechnological and pharmacological applications of toad and frog toxins**

Amphibians (toads, frogs, salamanders etc.) during their evolution have developed skin glands covering most parts of their body surface. From these glands small amounts of a mu‐ cous slime are secreted permanently, containing substances with different pharmacologic activities such as cardiotoxins, neurotoxins, hypotensive as well as hypertensive agents, he‐ molysins, and many others. Chemically they belong to a wide variety of substance classes such as steroids, alkaloids, indolalkylamines, catecholamines and low molecular peptides [11, 163]. Several studies have been showing new potential molecules for a variety of phar‐ macological applications from toads and frogs venoms.

### **5.1. Toxins acting on cardiovascular system**

type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas [156]. The antitumor activity of a potent antimicrobial peptide isolated from hemocytes of the spider *Acanthoscurria gomesiana*, named gomesin, was tested *in vitro* and *in vivo*. Gomesin showed cytotoxic and antitumor activities in cell lines, such as melano‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Several spider toxins have been studied as potential insecticidal bioactive with great biotech‐ nological possible applications [10]. A component of the venom of the Australian funnel web spider *Hadronyche versuta* that is a calcium channel antagonist retains its biological activity when expressed in a heterologous system. Transgenic expression of this toxin in tobacco effectively protected the plants from *Helicoverpa armigera* and *Spodoptera littoralis* larvae, with 100% mortality within 48h [158]. LiTxx1, LiTxx2 and LiTxx3 from *Loxosceles intermedia* venom were identified containing peptides that were active against *Spodoptera frugiperda*. These venom-derived products open a source of insecticide toxins that could be used as substitutes for chemical defensives and lead to a decrease in environmental prob‐ lems [159]. An insecticidal peptide referred to as Tx4(6-1) was purified from the venom of the spider *Phoneutria nigriventer* by a combination of gel filtration, reverse-phase fast liq‐ uid chromatography on Pep-RPC, reverse-phase high performance liquid chromatography (HPLC) on Vydac C18 and ion-exchange HPLC. The protein contains 48 amino acids includ‐ ing 10 Cys and 6 Lys. The results showed that Tx4(6-1) has no toxicity for mice, and sug‐ gest that it is a specific anti-insect toxin [160]. SMase D and homologs in the SicTox gene family are the most abundantly expressed toxic protein in venoms of *Loxosceles* and *Sicar‐ ius* spiders (Sicariidae). A recombinant SMase D from *Loxosceles arizonica* was obtained and compared its enzymatic and insecticidal activity to that of crude venom. SMase D and crude venom have comparable and high potency in immobilization assays on crickets. These da‐ ta indicate that SMase D is a potent insecticidal toxin, the role for which it presumably evolved [161]. δ-PaluIT1 and δ-paluIT2 are toxins purified from the venom of the spider *Paracoelotes luctuosus*. Similar in sequence to μ-agatoxins from *Agelenopsis aperta*, their phar‐ macological target is the voltage-gated insect sodium channel, of which they alter the inac‐ tivation properties in a way similar to α-scorpion toxins. Electrophysiological experiments on the cloned insect voltage-gated sodium channel heterologously co-expressed with the tipE subunit in *Xenopus laevis* oocytes, that δ-paluIT1 and δ-paluIT2 procure an increase of Na+ current [162]. Recently, several toxins have been isolated from spiders with potential

**5. Biotechnological and pharmacological applications of toad and frog**

Amphibians (toads, frogs, salamanders etc.) during their evolution have developed skin glands covering most parts of their body surface. From these glands small amounts of a mu‐

ma, breast cancer and colon carcinoma [157].

Applications

34

**4.7. Toxins with insecticides applications**

biotechnological application as insecticide.

**toxins**

Neurotensin-like peptides has been identified from frog skin, such as margaratensin, isolat‐ ed from *Rana margaratae* [164], a potential antihypertensive drug. Similar to the cardiac gly‐ cosides, bufadienolides from *Bufo bufo gargarizans* toad skin are able of inhibiting Na+ /K+ - ATPase, having an important role on treatment of congestive heart failure and arterial hypertension [165]. Examples of these bufadienolides are arenobufagin [166], cinobufagin, bufalin, resibufogenin, among others [165]. In the skin of *Rana temporaria* and *Rana igromacu‐ lata* frogs, bradykinin, a hypotensive and smooth muscle exciting substance, has been found [11]. Atelopidtoxin, a water-soluble toxin from skin of *Atelopus zeteki* frog, when injected into mammals, produces hypotension and ventricular fibrillation [167]. Semi-purified skin ex‐ tracts from *Pseudophryne coriacea* frog displayed effects on systemic blood pressure, reducing it by a probably cholinergic mechanism [168].

### **5.2. Toxins acting on hemostasis**

Annexins are a well-known multigene family of Ca2+-regulated membrane-binding and phospholipid-binding proteins. A novel annexin A2 (Bm-ANXA2) was isolated and purified from *Bombina maxima* skin homogenate, being the first annexin A2 protein reported to pos‐ sess platelet aggregation-inhibiting activity [169].

### **5.3. Toxins with antibiotic activity**

Toxins with antibiotic activity are the most well studied toxins in toads and frogs. Two anti‐ microbial bufadienolides, telocinobufagin and marinobufagin, were isolated from skin se‐ cretions of the Brazilian toad *Bufo rubescens* [170]. Antimicrobial peptides, named syphaxins (SPXs), were isolated from skin secretions of *Leptodactylus syphax* frog [171]. The alkaloids apinaceamine, 6-methyl-spinaceamine isolated from the skin gland secretions of *Leptodacty‐ lus pentadactylus* showed in screening tests bactericidal activity [172]. The cinobufacini and its active components bufalin and cinobufagin, from *Bufo bufo gargarizans* Cantor skin, pre‐ sented anti-hepatitis B virus (HBV) activity [173]. Telocinobufagin from *Rhinella jimi* toad were demonstrated to be active against *Leishmania chagasi* promastigotes and *Trypanosoma cruzi* trypomastigotes, while hellebrigenin, from same source, was active against only *T. cru‐ zi* trypomastigotes [174].

### **5.4. Toxins acting on inflammatory and nociceptive responses**

Epibatidine, an azabicycloheptane alkaloid isolated from the skin of frog *Epipedobates tricol‐ or*, was found to be a potent antinociceptive compound. Although its toxicity, this toxin could be a lead compound in the development of therapeutic agents for pain relief as well for treatment of disorders whose pathogenesis involves nicotinic receptors [175]. A variety of toxins acting on opioid receptors have been isolated from amphibians. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and related heptapeptide [Hyp6 ]-dermorphin isolated from the frog skin of *Phyllomedusa* sp., show higher affinity for μ-opioid receptors. Several peptides belonging to the dermorphin family have been isolated from frog skin [61]. Deltor‐ phins (also referred as dermenkephalin) and related peptides isolated from the frog skin have been found to exhibit high selectivity for δ-opiate receptors [176].

venom contains a variety of compounds peptides including melittin, apamin, adolapin, and mast cell degranulating (MCD) peptide, in addition of hyaluronidase and phospholipase A enzymes, that plays a variety of biological activities. The chemical constituents of venoms from wasps species include acetylcholine, serotonin, norepinephrine, hyaluronidase, histi‐ dine decarboxylase, phospholipase A2 and several polycationic peptides and proteins [12].

Toxins from Venomous Animals: Gene Cloning, Protein Expression and Biotechnological Applications

http://dx.doi.org/10.5772/52380

37

Honey bee venom and its main constituents have a marked effect on the cardiovascular system, most notably a fall in arterial blood pressure [183]. From the hemodynamic point of view, the venom, in higher doses, is extremely toxic to the circulatory system and in smaller doses, however, produce a stimulatory effect upon the heart [184]. Melittin, a strongly basic 26 aminoacid polypeptide which constitutes 40–60% of the whole dry honeybee venom, induces con‐ tractures and depolarization in skeletal muscle [12]. Melittin is cardiotoxic *in vitro*, causing arrest of the rat heart, but only induces a slight hypertension *in vivo* [183]. Apamin, without direct effect on contraction or relaxation, could attenuate the relaxation evoked by melittin at lower concentrations, and thus contribute to the conversion of melittin's relaxing activity into the contractile activity of the venom. Another peptide found in bee venom that outlines effects on the cardiovascular system is the Cardiopep. Cardiopep is a relatively nonlethal compo‐ nent, compared to phospholipase A, melittin, or whole bee venom itself. It is a potent nontox‐ ic beta-adrenergio-like stimulant that possesses definite anti-arrhythmic properties [185]. Studies on the cardiovascular effects of mastoparan B, isolated from the venom of the hor‐ net *Vespa basalis,* has shown that the peptide caused a dose-dependent inhibition of blood pressure and cardiac function in the rat. Research has shown that the cardiovascular effects of mastoparan B are mainly due to the actions of serotonin, and by a lesser extent to other

autacoids, released from mast cells as well from other biocompartments [186].

The mechanism by which bee venom affects the hemostatic system remains poorly under‐ stood [187]. Among the serine proteases isolated from bees, which acts as a fibrin(ogen)olyt‐ ic enzyme, activator prothrombin and directly degrades fibrinogen into fibrin degradation products, are the Bi-VSP (*Bombus ignitus*) [188], Bt-VSP (*Bombus terrestris*) [189] and Bs-VSP (*Bombus hypocrita sapporoensis*) [190]. According reference [188], the activation of prothrom‐ bin and fibrin(ogen)olytic activity may cooperate to effectively remove fibrinogen, and thus reduce the viscosity of blood. The injection fibrin(ogen)olytic enzyme can be used to facili‐ tate the propagation of components of bee venom throughout the bloodstream of mammals. Bumblebee venom also affects the hemostatic system through by Bi-KTI (*B. ignitus*), a Ku‐ nitz-type inhibitor, that strongly inhibited plasmin during fibrinolysis, indicating that Bi-KTI specifically targets plasmin [187]. A toxin protein named magnvesin was purified of *Vespa magnifica*. This protein contains serine protease-like activity inhibits blood coagulation, and was found to act on factors TF, VII, VIII, IX and X [191]. Other anticoagulant protein (protease I) with proteolytic activity was purified from *Vespa orientalis* venom, involving mainly coagulation factors VIII and IX [192]. Magnifin, a phospholipase A1 (PLA1) purified

**6.1. Toxins acting on cardiovascular system**

**6.2. Toxins acting on hemostasis**

#### **5.5. Toxins with anticancer and cytotoxic activities**

*Venenum Bufonis* is a traditional Chinese medicine obtained from the dried white secretion of auricular and skin glands of Chinese toads (*Bufo melanostictus* Schneider or *Bufo bufo gargar‐ zinas* Cantor). Cinobufagin (CBG), isolated from *Venenum Bufonis*, had potential immune system regulatory effects and is suggested that this compound could be developed as a nov‐ el immunotherapeutic agent to treat immune-mediated diseases such as cancer [177]. Bufa‐ dienolides from toxic glands of toads are used as anticancer agents, mainly on leukemia cells. Bufalin and cinobufagin from *Bufo bufo gargarizans* Cantor were tested and studies shown that these toxins suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators [178]. Bufotalin, one of the bufadienolides iso‐ lated from Formosan Ch'an Su, which is made of the skin and parotid glands of toads, in‐ duce apoptosis in human hepatocellular carcinoma, probably involving caspases and apopotosis-inducing factor [179]. Cutaneous venom of *Bombina variegata pachypus* toad pre‐ sented a cytolitic effect on the growth of the human HL 60 cell line [180]. Brevinin-2R, a nonhemolytic defensin has been isolated from the skin of the frog *Rana ridibunda*, showing pronounced cytotoxicity towards malignant cells [181].

#### **5.6. Toxins with insulin releasing activity**

Diabetes mellitus is a disease in which the body is unable to sufficiently produce or properly use insulin. Newer therapeutic modalities for this disease are extremely needed. Peptides with insulin-releasing activity have been isolated from the skin secretions of the frog *Aga‐ lychnis litodryas* and may serve as templates for a novel class of insulin secretagogues [182].
