**4. Discussion**

576 Pharmacology

Figure 4 shows the effect of commercial 0.05 mg/mL theaflavin and 0.025 mg/mL epigallocatechin gallate from *C. sinensis* on twitch blockade induced by 40 µg/mL Bjssu venom. This paralysis was completely blocked (n=3, \*p<0.05 compared to the venom, but did not show statistical differences with *C. sinensis* extract). In addition, following washing out of treated preparation with fresh physiological salt solution, twitch height was re-

Bjssu 40 µg/mL + theaflavin 0.05 mg/mL (n=3)

Bjssu 40 µg/mL + epigallocathechin gallate 0.025 mg/mL (n=3)

0 20 40 60 80 100 120

Fig. 4. Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Antibothropic action of commercial phytochemicals from Camellia sinensis. Note total protection against the paralysis of Bjssu (Bothrops jararacussu) venom. Each point represents the mean ± SEM. \* = p<0.05 in comparison with the crude venom.

Figure 5 shows a chromatoplaque of *C. sinensis* leaves extract obtained by TLC exhibiting a complex variety of compounds including theaflavin and epigallocatechin as confirmed by Rf of these commercial phytochemicals. Panel A is the chromatoplaque exposed only to a UV light at 360 nm, whereas Panel B is the same plaque after NP/PEG chromogenic agent

Time (min)

\* \* \* \* \* \* \* \* \*

**3.3 Efficacy of commercial phytochemicals against Bjssu venom** 

Bjssu 40 µg/mL (n=5)

established (not shown).

0

pulverization.

**3.4 Thin layer chromatography (TLC)** 

20

40

60

Twitch tension (%)

80

\*

\*

100

Although the only specific treatment for envenoming by snakebites is immunoglobulins (antivenoms), since it can prevent or reverse most of the systemic effects and hence minimizing mortality and morbidity (WHO, 2011a), any alternative strategy aiming to interrupt or neutralize the steps of envenoming process can be effective for snakebite local effects. The clinical features of the bites of venomous snakes reflect the effects of these venom components that vary between species to species, but can broadly be divided into categories which include i) cytotoxins, causing local swelling and tissue damage, ii) haemorrhagins, which disturb the integrity of blood vessels, iii) compounds, which lead to incoagulable blood, iv) neurotoxins, causing neurotoxicity and iv) myotoxins, which cause muscle breakdown (WHO, 2011b). *Bothrops jararacussu* venom encloses all of them, except *in vivo* neurotoxicity (Milani et al*.*, 1997), but it causes an *in vitro* neuromuscular blockade (Rodrigues-Simioni et al*.*, 1983).

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

signaling.

neuromuscular junction.

about these mechanisms.

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 579

partial muscular activity in the presence of 5 μg/mL venom. Coincidently, Basu et al. (2005) showed that only the theaflavin fraction from black tea was able to produce a facilitatory effect at the skeletomotor site, being this facilitation modulated by calcium and nitric oxide

Concerning the modulation of synaptic nerve-muscle interaction, it was found that ACh and glutamate are co-released from synaptosomes of *Torpedo electric* organ (Vyas & Bradford, 1987), also demonstrated in rat motor nerve terminals (Waerhaug & Ottersen, 1993). Glutamatergic receptors such as N-methyl-D-aspartate (NMDA) have been identified at the postsynaptic membrane in neuromuscular junction of adult rats (Urazaev et al., 1998; Grozdanovic & Gossrau, 1998). Glutamate released from nerve endings probably activates NMDA-receptor mediated Ca2+ entry into the sarcoplasm followed by activation of NO (Urazaev et al., 1998). Nonquantal ACh acting through M1-cholinergic receptors (Urazaev et al., 2000; Malomouzh et al., 2007), activates synthesis of NO to serve as a trophic message from motoneurones that keeps the Cl- transport inactive in the innervated sarcolemma (Urazaev et al., 1999). For a better understanding of the synaptic nerve-muscle modulation, see also the study of Rubem-Mauro et al. (2009) that corroborates the nitric oxide role at the

 Fig. 6. Structures of major components of *Camellia sinensis*. A, epigallocatechin gallate (Zhu

**A B** 

The well-successful experience between *C. sinensis* leaf extract and presynaptic neurotoxins, and while bothropstoxin-I (BthTX-I) isolated from *Bothrops jararacussu* venom exhibits an earlier presynaptic action (Oshima-Franco et al., 2004) before its well-known myotoxic effect, the same experimental procedure was carried out using *C. sinensis* extract, which

Different mechanisms have been proposed for BthTX-I myotoxic effect such as altering the bilayer membrane integrity (Lomonte et al., 2003), binding to the Ca2+ -binding region in the pore of Ca2+ channels (Oshima-Franco et al*.*, 2004), activating membrane acceptors (Cintra-Francischinelli et al*.*, 2009) or causing a general membrane-destabilizing (Gallacci & Cavalcante, 2009). In order to explain the rationale of this study more details will be given

et al., 2008). B, theaflavin (Khan & Mukhtar, 2007).

protected 100% the neuromuscular blockade (Fig. 1).

As snake accidents occur by bites and venoms are commonly injected in the subcutaneous muscle tissue, the use of muscle preparations as model for the study of the pharmacological effects of snake venom and toxins is very relevant. Besides, the use of snake venom and toxins as tools to study neuromuscular blockade *in vitro* (Gallacci & Cavalcante, 2010) is very useful given the excitation-contraction coupling process starts with transmission of electrical impulses from nerves towards muscle fibers via release of acetylcholine (ACh) (Hughes et al., 2006).

On the other hand, the plant kingdom represents a rich resource of new molecules able to counteract the venom effects, mainly when the plant is as worldwide as *Camellia sinensis*, an evergreen Asiatic shrub of the Theaceae family. Polyphenols from black or green tea has been shown to be powerful antioxidants with a potent inhibitory effect on low density lipoprotein (LDL) oxidation *in vitro* (Miura et al*.*, 2000), exert anti-carcinogenic (Lambert & Yang, 2003) and anti-inflammatory (Arab & Il'yasova, 2003) effects; act as antibacterial and antiviral agents (Friedman, 2007), and are able to reduce the incidence of coronary heart disease and diabetes (Crozier et al*.*, 2009), among other effects (see Khan & Mukhtar, 2007). Despite its health benefits, there are few studies using *C. sinensis* addressed to snake venom.

Hung et al. (2004) showed an antagonistic effect of 3 mg per mouse of melanin extracted from black tea (MEBT), an unhydrolyzed complex of tea polyphenols (Sava et al*.*, 2001), against *Agkistrodon contortrix laticinctus* (broadbanded copperhead), *Agkistrodon halys blomhoffii* (Japanese mamushi), and *Crotalus atrox* (western diamondback rattlesnake) snake venoms, when administered i.p. immediately after venom administration in the same place of venom injection. Authors demonstrated correlation between antivenom activity of melanin and PLA2 inhibition as a possible explanation for the protective effect.

Tea polyphenols have been shown to interact with hydrolytic enzymes from *Naja naja kaouthia* Lesson (Elapidae) and *Calloselasma rhodostoma* Kuhl (Viperidae) venoms, inhibiting inflammation and local tissue damage. This effect was attributed to complexation and chelation among the venom proteins and the phenolic contents of the extract. According to the authors, the *Camellia sinensis* extract also inhibited phospholipase A2, proteases, hyaluronidase and L-amino acid oxidase by *in vitro* neutralization and the hemorrhagic and the dermonecrotic activities of the venoms *in vivo* (Pithayanukul et al*.*, 2010).

Satoh et al. (2002 a,b) reported the protective effect of thearubigin from black tea extract against the neuromuscular blockade caused by botulin neurotoxins and tetanus toxin in synaptosomal membrane preparations. Recently, de Jesus Reis Rosa et al. (2010) reiterate the protective effect of C. sinensis leaves extracts which prevented in vitro the irreversible neuromuscular blockade typical of Crotalus durissus terrificus venom, more specifically caused by crotoxin, the main component of the crude venom (Slotta & Fraenkel-Conrat, 1983). We suggest that the target for C. sinensis protective effect is the motor nerve terminal, since the blockade caused by crotoxin, botulin toxin and tetanus toxin occurs by the inhibition of the neurotransmitter release, differently, from motor nerve terminals (Habermann et al., 1980).

Based on research findings suggesting an effective anti-cancer property attributed mainly to epigallocathechin-3-gallate (Fig. 6A) found primarily in green tea, and theaflavin (Fig. 6B) from black tea, both equally effective antioxidants (Leung et al., 2001), these two compounds were also assayed against *Crotalus durissus terrificus* venom (de Jesus Reis Rosa et al., 2010). Curiously, commercial theaflavin, but not epigallocathechin gallate, maintained

As snake accidents occur by bites and venoms are commonly injected in the subcutaneous muscle tissue, the use of muscle preparations as model for the study of the pharmacological effects of snake venom and toxins is very relevant. Besides, the use of snake venom and toxins as tools to study neuromuscular blockade *in vitro* (Gallacci & Cavalcante, 2010) is very useful given the excitation-contraction coupling process starts with transmission of electrical impulses from nerves towards muscle fibers via release of acetylcholine (ACh)

On the other hand, the plant kingdom represents a rich resource of new molecules able to counteract the venom effects, mainly when the plant is as worldwide as *Camellia sinensis*, an evergreen Asiatic shrub of the Theaceae family. Polyphenols from black or green tea has been shown to be powerful antioxidants with a potent inhibitory effect on low density lipoprotein (LDL) oxidation *in vitro* (Miura et al*.*, 2000), exert anti-carcinogenic (Lambert & Yang, 2003) and anti-inflammatory (Arab & Il'yasova, 2003) effects; act as antibacterial and antiviral agents (Friedman, 2007), and are able to reduce the incidence of coronary heart disease and diabetes (Crozier et al*.*, 2009), among other effects (see Khan & Mukhtar, 2007). Despite its health benefits, there are few studies using *C. sinensis* addressed to snake venom. Hung et al. (2004) showed an antagonistic effect of 3 mg per mouse of melanin extracted from black tea (MEBT), an unhydrolyzed complex of tea polyphenols (Sava et al*.*, 2001), against *Agkistrodon contortrix laticinctus* (broadbanded copperhead), *Agkistrodon halys blomhoffii* (Japanese mamushi), and *Crotalus atrox* (western diamondback rattlesnake) snake venoms, when administered i.p. immediately after venom administration in the same place of venom injection. Authors demonstrated correlation between antivenom activity of

melanin and PLA2 inhibition as a possible explanation for the protective effect.

the dermonecrotic activities of the venoms *in vivo* (Pithayanukul et al*.*, 2010).

Tea polyphenols have been shown to interact with hydrolytic enzymes from *Naja naja kaouthia* Lesson (Elapidae) and *Calloselasma rhodostoma* Kuhl (Viperidae) venoms, inhibiting inflammation and local tissue damage. This effect was attributed to complexation and chelation among the venom proteins and the phenolic contents of the extract. According to the authors, the *Camellia sinensis* extract also inhibited phospholipase A2, proteases, hyaluronidase and L-amino acid oxidase by *in vitro* neutralization and the hemorrhagic and

Satoh et al. (2002 a,b) reported the protective effect of thearubigin from black tea extract against the neuromuscular blockade caused by botulin neurotoxins and tetanus toxin in synaptosomal membrane preparations. Recently, de Jesus Reis Rosa et al. (2010) reiterate the protective effect of C. sinensis leaves extracts which prevented in vitro the irreversible neuromuscular blockade typical of Crotalus durissus terrificus venom, more specifically caused by crotoxin, the main component of the crude venom (Slotta & Fraenkel-Conrat, 1983). We suggest that the target for C. sinensis protective effect is the motor nerve terminal, since the blockade caused by crotoxin, botulin toxin and tetanus toxin occurs by the inhibition of the neurotransmitter release, differently, from motor nerve terminals (Habermann et al., 1980).

Based on research findings suggesting an effective anti-cancer property attributed mainly to epigallocathechin-3-gallate (Fig. 6A) found primarily in green tea, and theaflavin (Fig. 6B) from black tea, both equally effective antioxidants (Leung et al., 2001), these two compounds were also assayed against *Crotalus durissus terrificus* venom (de Jesus Reis Rosa et al., 2010). Curiously, commercial theaflavin, but not epigallocathechin gallate, maintained

(Hughes et al., 2006).

partial muscular activity in the presence of 5 μg/mL venom. Coincidently, Basu et al. (2005) showed that only the theaflavin fraction from black tea was able to produce a facilitatory effect at the skeletomotor site, being this facilitation modulated by calcium and nitric oxide signaling.

Concerning the modulation of synaptic nerve-muscle interaction, it was found that ACh and glutamate are co-released from synaptosomes of *Torpedo electric* organ (Vyas & Bradford, 1987), also demonstrated in rat motor nerve terminals (Waerhaug & Ottersen, 1993). Glutamatergic receptors such as N-methyl-D-aspartate (NMDA) have been identified at the postsynaptic membrane in neuromuscular junction of adult rats (Urazaev et al., 1998; Grozdanovic & Gossrau, 1998). Glutamate released from nerve endings probably activates NMDA-receptor mediated Ca2+ entry into the sarcoplasm followed by activation of NO (Urazaev et al., 1998). Nonquantal ACh acting through M1-cholinergic receptors (Urazaev et al., 2000; Malomouzh et al., 2007), activates synthesis of NO to serve as a trophic message from motoneurones that keeps the Cl- transport inactive in the innervated sarcolemma (Urazaev et al., 1999). For a better understanding of the synaptic nerve-muscle modulation, see also the study of Rubem-Mauro et al. (2009) that corroborates the nitric oxide role at the neuromuscular junction.

Fig. 6. Structures of major components of *Camellia sinensis*. A, epigallocatechin gallate (Zhu et al., 2008). B, theaflavin (Khan & Mukhtar, 2007).

The well-successful experience between *C. sinensis* leaf extract and presynaptic neurotoxins, and while bothropstoxin-I (BthTX-I) isolated from *Bothrops jararacussu* venom exhibits an earlier presynaptic action (Oshima-Franco et al., 2004) before its well-known myotoxic effect, the same experimental procedure was carried out using *C. sinensis* extract, which protected 100% the neuromuscular blockade (Fig. 1).

Different mechanisms have been proposed for BthTX-I myotoxic effect such as altering the bilayer membrane integrity (Lomonte et al., 2003), binding to the Ca2+ -binding region in the pore of Ca2+ channels (Oshima-Franco et al*.*, 2004), activating membrane acceptors (Cintra-Francischinelli et al*.*, 2009) or causing a general membrane-destabilizing (Gallacci & Cavalcante, 2009). In order to explain the rationale of this study more details will be given about these mechanisms.

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

calcium modulating factor is also other possibility.

epigallocatechin gallate is also interesting.

snake venoms, a question that remains to be cleared.

*sinensis* extract and the BthTX-I interaction remains to be cleared.

neurotoxicity and myotoxicity induced by the venom and the myotoxin.

2010).

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 581

Fig. 7. Molecular structure of snake venom Lys49 PLA2 homologue (Gallacci & Cavalcante,

Basu et al. (2005) showed that the theaflavins-induced facilitation was dependent on the calcium concentration of the physiological solution pointing to an involvement of calcium in the facilitatory action of theaflavins. It is evident that the skeletal muscle can contract in the absence of external calcium, but under physiological conditions, when calcium is present in the medium, it induces the release of stored calcium from the sarcoplasmic reticulum in order to maintain the optimal integrity of the contractile mechanism (Endo, 1985). Considering that *C. sinensis* extract totally prevent the neuromuscular blockade induced by the myotoxin and calcium seems to be involved in the toxic mechanism of BthTX-I, by different proposed mechanisms as already discussed, the explanation of Basu et al*.* (2005) that *C. sinensis*, produces a facilitatory effect, via theaflavin, acting presynaptically as

In spite of the hypothesis discussed here, the actual molecular mechanism involving the *C.* 

Here, when the efficacy of *C. sinensis* extract was assayed against the crude venom, 80% of the contractile response was found preserved even after two hours of the venom exposure (Fig. 2), a promising result, since venom has a complex composition, differently from BthTX-I. Histological analyses clearly showed the protective effect of *C. sinensis* extract against the myotoxic action of venom (Fig. 3E), showing the same positive correlation between

Whereas only the commercial theaflavin protected against the neuromuscular blockade of *Crotalus durissus terrificus* (de Jesus Reis Rosa et al*.*, 2010), here, both theaflavin and epigallocatechin gallate, totally protected against the paralysis by *Bothrops jararacussu* venom (Fig. 4), a result better than that produced by *C. sinensis* extract, since the amount of these phytochemicals in the extract (as shown in Fig. 5) is lesser than that used in the neutralization assays. However, the *C. sinensis* extract contains a multitude of other compounds, which real participation against the toxic effects of venom must be assayed, hence using an *in vivo* model simulating the cronically black or green tea consumption (by humans) followed by subcutaneous injection of the venoms. A comparison between the treatment with commercial antivenom alone and commercial antivenom plus theaflavin or

It is well-known that *C. durissus terrificus* and *B. jararacussu* venoms act differently in inducing clinical symptoms as well as *in vitro* paralysis at skeletomotor apparatus. Considering that venoms were previously incubated with each commercial phytochemical, and that epigallocatechin gallate, the major catechin in green tea, totally inhibited the toxic compounds of *B. jararacussu* venom, but did not do so against the rattlesnake venom, it is reasonable to suggest that theaflavin inhibits both presynaptic and postsynaptic venom effects, whereas epigallocatechin gallate inhibits mainly postsynaptic venom effects of these

BthTX-I represents a distinct group of PLA2 homologue myotoxins containing Lys49 instead of Asp49 residue, with consequent loss of Ca2+-binding and enzymatic activity, the segment 115-129 of the C-terminal region, which includes a variable combination of positively charged and hydrophobic/aromatic residues, has the ability to alter the bilayer membrane integrity (Lomonte et al., 2003), a possible way by which *C. sinensis* extract could exert its protection against the toxic effect of BthTX-I.

Oshima-Franco et al. (2004) have shown that BthTX-I, at a concentration that does not produce neuromuscular blockade (0.35 mM) caused the appearance of giant miniature endplate potentials, without affecting the resting membrane potential. The authors suggested that the toxin would act through Ca2+ channels, since Mn2+ antagonized both neurotoxic and myotoxic actions of the myotoxin and are related to Ca2+fluxes. Mn2+ is thought to bind to the Ca2+ -binding region in the pore of Ca2+ channels, thereby preventing the passage of calcium ions (Nachshen, 1984). The influence of the earlier presynaptic action of BthTX-I is relevant from the pharmacological point of view, as shown here using *C. sinensis* leaves extract, although clinically the bothropic envenomation shows no signs of neurotoxicity. However, *C. sinensis* extract also protected against the myotoxic effects of BthTX-I (Fig. 3D), showing a parallelism between neurotoxic and myotoxic effects of the myotoxin.

Cintra-Francischinelli et al. (2009) excluded the possibility that the inactive Lys49 toxins act by binding to a membrane channel, thus increasing its permeability to Ca2+. The authors have shown that the action of myotoxins from snake venoms on muscle cells begins with the activation of membrane acceptors coupled to intracellular Ca2+ stores, which is rapidly followed by the toxin dependent alteration of membrane permeability to ions (and other molecules). By this mechanism, *C. sinensis* is able to inactivate the acceptors signalization.

Gallacci & Cavalcante (2010) proposed a hypothetical mechanism for the *in vitro* neuromuscular blockade induced by snake venom Lys49 PLA2 homologues (Fig. 7): the binding of the Lys49 PLA2 homologues to hydrophobic domains in muscle plasma membrane promotes a non-enzymatic alteration of the membrane structure. As a consequence, there is a colapse of the ionic gradient and depolarization of both muscle fiber and nerve terminal, mainly due to re-equilibration of sodium and potassium ions concentration. The persistent cell depolarization could inactivate voltage-dependent sodium channels in the perijunctional zone. Consequently, the threshold of excitability of the muscle fiber rises out of the reach of the endplate potential; no action potential is triggered and the neuromuscular transmission is blocked. The depolarization of nerve terminal could increase the spontaneous release of acetylcholine, i.e. the frequency of miniature endplate potentials. The action potentials superimposed on the background level of nerve depolarization are reduced since the membrane potential is already shifted nearer to the sodium equilibrium potential. The reduced action potentials promote a decreased calcium influx and consequently a reduction of releasing of evoked acetylcholine. The muscle fiber membrane disruption induced by Lys49 PLA2 homologues also promotes an increase in the concentration of cytosolic calcium that initiates a complex series of degenerative effects on muscle fiber. By this mechanism, *C. sinensis* extract efficiently did avoid the initial trigger.

BthTX-I represents a distinct group of PLA2 homologue myotoxins containing Lys49 instead of Asp49 residue, with consequent loss of Ca2+-binding and enzymatic activity, the segment 115-129 of the C-terminal region, which includes a variable combination of positively charged and hydrophobic/aromatic residues, has the ability to alter the bilayer membrane integrity (Lomonte et al., 2003), a possible way by which *C. sinensis* extract could exert its

Oshima-Franco et al. (2004) have shown that BthTX-I, at a concentration that does not produce neuromuscular blockade (0.35 mM) caused the appearance of giant miniature endplate potentials, without affecting the resting membrane potential. The authors suggested that the toxin would act through Ca2+ channels, since Mn2+ antagonized both neurotoxic and myotoxic actions of the myotoxin and are related to Ca2+fluxes. Mn2+ is thought to bind to the Ca2+ -binding region in the pore of Ca2+ channels, thereby preventing the passage of calcium ions (Nachshen, 1984). The influence of the earlier presynaptic action of BthTX-I is relevant from the pharmacological point of view, as shown here using *C. sinensis* leaves extract, although clinically the bothropic envenomation shows no signs of neurotoxicity. However, *C. sinensis* extract also protected against the myotoxic effects of BthTX-I (Fig. 3D), showing a parallelism between

Cintra-Francischinelli et al. (2009) excluded the possibility that the inactive Lys49 toxins act by binding to a membrane channel, thus increasing its permeability to Ca2+. The authors have shown that the action of myotoxins from snake venoms on muscle cells begins with the activation of membrane acceptors coupled to intracellular Ca2+ stores, which is rapidly followed by the toxin dependent alteration of membrane permeability to ions (and other molecules). By this mechanism, *C. sinensis* is able to inactivate the

Gallacci & Cavalcante (2010) proposed a hypothetical mechanism for the *in vitro* neuromuscular blockade induced by snake venom Lys49 PLA2 homologues (Fig. 7): the binding of the Lys49 PLA2 homologues to hydrophobic domains in muscle plasma membrane promotes a non-enzymatic alteration of the membrane structure. As a consequence, there is a colapse of the ionic gradient and depolarization of both muscle fiber and nerve terminal, mainly due to re-equilibration of sodium and potassium ions concentration. The persistent cell depolarization could inactivate voltage-dependent sodium channels in the perijunctional zone. Consequently, the threshold of excitability of the muscle fiber rises out of the reach of the endplate potential; no action potential is triggered and the neuromuscular transmission is blocked. The depolarization of nerve terminal could increase the spontaneous release of acetylcholine, i.e. the frequency of miniature endplate potentials. The action potentials superimposed on the background level of nerve depolarization are reduced since the membrane potential is already shifted nearer to the sodium equilibrium potential. The reduced action potentials promote a decreased calcium influx and consequently a reduction of releasing of evoked acetylcholine. The muscle fiber membrane disruption induced by Lys49 PLA2 homologues also promotes an increase in the concentration of cytosolic calcium that initiates a complex series of degenerative effects on muscle fiber. By this mechanism, *C. sinensis*

protection against the toxic effect of BthTX-I.

neurotoxic and myotoxic effects of the myotoxin.

extract efficiently did avoid the initial trigger.

acceptors signalization.

Fig. 7. Molecular structure of snake venom Lys49 PLA2 homologue (Gallacci & Cavalcante, 2010).

Basu et al. (2005) showed that the theaflavins-induced facilitation was dependent on the calcium concentration of the physiological solution pointing to an involvement of calcium in the facilitatory action of theaflavins. It is evident that the skeletal muscle can contract in the absence of external calcium, but under physiological conditions, when calcium is present in the medium, it induces the release of stored calcium from the sarcoplasmic reticulum in order to maintain the optimal integrity of the contractile mechanism (Endo, 1985). Considering that *C. sinensis* extract totally prevent the neuromuscular blockade induced by the myotoxin and calcium seems to be involved in the toxic mechanism of BthTX-I, by different proposed mechanisms as already discussed, the explanation of Basu et al*.* (2005) that *C. sinensis*, produces a facilitatory effect, via theaflavin, acting presynaptically as calcium modulating factor is also other possibility.

In spite of the hypothesis discussed here, the actual molecular mechanism involving the *C. sinensis* extract and the BthTX-I interaction remains to be cleared.

Here, when the efficacy of *C. sinensis* extract was assayed against the crude venom, 80% of the contractile response was found preserved even after two hours of the venom exposure (Fig. 2), a promising result, since venom has a complex composition, differently from BthTX-I. Histological analyses clearly showed the protective effect of *C. sinensis* extract against the myotoxic action of venom (Fig. 3E), showing the same positive correlation between neurotoxicity and myotoxicity induced by the venom and the myotoxin.

Whereas only the commercial theaflavin protected against the neuromuscular blockade of *Crotalus durissus terrificus* (de Jesus Reis Rosa et al*.*, 2010), here, both theaflavin and epigallocatechin gallate, totally protected against the paralysis by *Bothrops jararacussu* venom (Fig. 4), a result better than that produced by *C. sinensis* extract, since the amount of these phytochemicals in the extract (as shown in Fig. 5) is lesser than that used in the neutralization assays. However, the *C. sinensis* extract contains a multitude of other compounds, which real participation against the toxic effects of venom must be assayed, hence using an *in vivo* model simulating the cronically black or green tea consumption (by humans) followed by subcutaneous injection of the venoms. A comparison between the treatment with commercial antivenom alone and commercial antivenom plus theaflavin or epigallocatechin gallate is also interesting.

It is well-known that *C. durissus terrificus* and *B. jararacussu* venoms act differently in inducing clinical symptoms as well as *in vitro* paralysis at skeletomotor apparatus. Considering that venoms were previously incubated with each commercial phytochemical, and that epigallocatechin gallate, the major catechin in green tea, totally inhibited the toxic compounds of *B. jararacussu* venom, but did not do so against the rattlesnake venom, it is reasonable to suggest that theaflavin inhibits both presynaptic and postsynaptic venom effects, whereas epigallocatechin gallate inhibits mainly postsynaptic venom effects of these snake venoms, a question that remains to be cleared.

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

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#### **5. Conclusion**

*Camellia sinensis* leaves extract possesses inhibitory effect against the neuromuscular blockade induced by *Bothrops jararacussu* venom and also bothropstoxin-I, by an unclear mechanism of action. Altogether, the data suggest that theaflavin and epigallocatechin gallate have a strong participation on these protective effects.

#### **6. Acknowledgment**

This work was supported by a research grant from São Paulo Research Foundation FAPESP, Proc. 04/09705-8, 07/53883-6, 08/52643-4. L.J.R.R. is a student of Post-Graduation Course in Pharmaceutical Sciences (Master level) from UNISO. G.A.A.S. had a scholarship (I.C.) from PROBIC/UNISO.

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**26** 

Eva Kovacs

*Switzerland* 

*Cancer Immunology Research* 

**The Effects of** *Viscum album*

**Several and Different Parameters** 

**(Mistletoe) QuFrF Extract and Vincristine** 

**in Human Multiple Myeloma Cell Lines –** 

**A Comparative Experimental Study Using** 

**Multiple myeloma** (MM) is a haematological disorder of malignant plasma cells. B

When they are activated to secrete antibodies, they are known as plasma cells, which are crucial part of the immune system. Due to the fundamental nature of the system affected, multiple myeloma manifests systemic symptoms that make it difficult to diagnose. Multiple myeloma is characterised by slow proliferation of the tumour cells, mainly in the bone marrow, by production of large amounts of immunoglobulins and osteolytic lesions. Multiple myeloma is a generally incurable disease at present, but remissions may be induced with stem cells transplants, steroids, chemotherapy and treatment with vincristine + doxorubicin + dexamethasone or thalidomide + dexamethasone or bortezomib based regimens or

Multiple myeloma mainly affects older adults, but its causes and other risk factors are unknown. Yearly incidence is 3-6/100 000 worldwide, accounts for 1-2 % of all human

**1.3 The role of cytokines in the growth, progression and dissemination of multiple** 

**Cytokines** are soluble proteins, peptides or glycoproteins that are released by cells. Cytokines can affect the cells via an autocrine and/or paracrine regulation mechanisms. In case of an autocrine regulation mechanism the endogenous produced cytokine affects the same type of cell. In case of a paracrine regulation mechanism the target cell is near to the

lenalidomide. The different therapeutic modalities have different "target location".

lymphocytes start in the bone marrow and move to the lymph nodes.

**1. Introduction** 

**myeloma** 

**1.1 Multiple myeloma** 

**1.2 Epidemiology of multiple myeloma** 

cancer. Median survival is 50–55 months.

G. Gosmann, J.C.P. Mello, L.A. Mentz, & P.R. Petrovick, (Eds.), UFRGS/UFSC, ISBN 85-7025-682-5, Porto Alegre/Florianópolis, Brazil


<http://www.who.int/neglected\_diseases/diseases/snakebites/en/>.

