*2.4. Statistical Models*

In this work, we used an ensemble approach (biomod2) [22] to describe the current and future habitat of *Euacalyptus grandis* and *E. dunnii* in Uruguay, as a basis to study potential changes in the optimal plantation areas for both species, under different climate scenarios. The biomod2 R package assembles the SDM which includes the Generalized Lineal Model (GLM), Generalized Additive Models (GAM), classification and regression trees (CART), Boosted Regression Tress (BRT), Random Forest (RF), Maximum entropy (MaxEnt) flexible determinant analysis (FDA), artificial neuronal networks (ANN), Multivariate Adaptive Regression Splines (MARS), and Surface Range Envelope (SRE). We used ensemble models calculated using the mean, median, coefficient of variation, confidence interval (inferior and superior), committee averaging (CA), and probability mean weight decay (WD) of the single model prediction. The CA was achieved by a binary (presence/absence) transformation using the threshold of the single model predictions. The threshold is the maximum score of the evaluation metric ("True Skill Statistics", TSS) for the evaluated dataset. Subsequently, the probability value of each pixel was calculated as the mean of the single pixel predictions. The WD ensemble modelling scaled the individual model predictions according to their accuracy statistic value (AUC) and the sum of all individual models [37,40]. We made ensemble predictions based on all single models' projections with an AUC > 0.90.

#### *2.5. Selection and Validation of the Model*

The evaluation and selection of the model correspond to the determination of the predictive capacity based on the quantification of the error and the misclassified data. These errors may be of commission, which is the classification as an absence of a data point that is present (false negative), and of omission, which is the opposite. We randomly split our dataset into two subsets, 70% of the data to train the model and 30% for model evaluation using cross-validation (100) which yielded 100 different fits per model. Models with higher mean values and smaller variations were considered as being the most accurate ones [28]. The criteria used to determine the predictive capacity of the

model were the Kappa coefficient (K), the Area Under the ROC (Operational Characteristic of the Receptor) Curve (AUC) and the coefficient TSS. The first of these was used to estimate the veracity of the maps developed and is a qualitative measure of the concordance between the categoric predictors and describes the concordance rate between the observed and predicted values. The values of K vary from +1 to −1, where +1 represents a perfect classification and negative values represent a random fitment [41]. The AUC is the graphical representation of the commission errors on the horizontal axis (presences classified incorrectly, 1-specificity), and the omission errors (real presences which are omitted, sensitivity) are represented on the vertical axis for the whole value range [42]. The values obtained vary from 0.5 to 1, where 1 represents a perfect classification (presences and absences) and 0.5 represents a random prediction. According to Thuiller et al. [43], the fitted model may be classified as poor (AUC < 0.8), satisfactory (0.8 < AUC < 0.9), good (0.9 < AUC < 0.95), or very good (0.95 < AUC < 1). The coefficient TSS was applied to estimate the precision of the model with the presence/pseudo-absence data [44]. This compares the number of correct predictions except for those attributed to the random and, in the same way as the K index, considers the omission and commission errors. Its values oscillate between −1 and 1, where 1 indicates a perfect concordance and negative values indicate random behavior [42]. This index is defined according to the following expression:

$$\text{TSS} = \text{sensitivity} + \text{specificity} - 1 \tag{2}$$

In this case, K index and TSS were calculated according to maximum TSS threshold [45].

#### *2.6. Comparison of Current and Future Distributions*

Current and future model predictions were obtained at a pixel resolution ~1 km. Each pixel of the prediction contains the probability of occurrence of the species ranging from 0 (not probable) to 1 (highly probable). To better visualization of the probability of occurrence the probability values were classified in four categories (0–25%, low; 26–50%, moderately low; 51–75%, moderately high; 76–100%, high) for the present or future occurrence of both species in Uruguay. To calculate the variation in the area occupied in the future, compared to the area currently occupied by the two species, the probability maps were reclassified to 0 (absence) and 1 (present), using the same threshold applied to calculate TSS and Kappa. The total area of the potential distribution for each single projection was calculated by counting the number of pixels and multiplies it by the area of a pixel and the changed area was presented as percentage of total area loss (<100%) or gained (>100%) [30].

#### **3. Results**


**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

No.5, (October 1994), pp. 470–471, ISSN 0032-0943

No.3, (August 1997), pp. 197–201, ISSN 0378-8741

(January 2007), pp. 116-134, ISSN 1613-4125

(October 2009), pp. 1–11, ISSN 0041-0101

(December 2009), pp. e569-e575, ISSN 1935-2735

pp.57-63, ISSN 0302-766X

217-229, ISSN 0014-4800

3431S-3440S, ISSN 1541-6100

2009), pp. 1001–1043, ISSN 0265-0568

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 583

Crespy, V. & Williamson, G. (2004). A review of the health effects of green tea catechins *in*

Crozier, A., Jaganath, I.B. & Clifford, M.N. (2009). Dietary phenolics: chemistry,

Das, M., Vedasiromoni, J.R., Chauhan, S.P.S. & Ganguly, D.K. (1994). Effect of the hot-water

Das, M., Vedasiromoni, J.R., Chauhan, S.P.S. & Ganguly, D.K. (1997). Effect of green tea

De Jesus Reis Rosa, L., Silva, G.A.A., Amaral Filho, J., Silva, M.G., Cogo, J.C., Groppo, F.C. &

dos Santos, J.I., Cintra-Francischinelli, M., Borges, R.J., Fernandes, C.A., Pizzo, P., Cintra,

A₂ class. *Proteins,* Vol.79, No.1, (September 2010), pp. 61-78, ISSN 0887-3585 Endo, M. (1985). Calcium release from sarcoplasmic reticulum. In: Bronner, F. (Ed.), *Current Topics in Membrane and Transport*, 25, Academic Press, London, pp. 181-230 Fenwick, A.M., Gutberlet-Jr, R.L., Evans, J. & Parkinson, C.L. (2009). Morphological and

Friedman, M. (2007). Overview of antibacterial, antitoxin, antiviral, and antifungal activities

Gallacci, M. & Cavalcante, W.L.G. (2010). Understanding the in vitro neuromuscular activity

Graham, H. N (1992). Green tea composition, consumption, and polyphenol chemistry. *Preventive Medicine*, Vol.21, No.3, (May 1992), pp. 334-350, ISSN 0091-7435 Grozdanovic, Z. & Gossrau, R. (1998). Co-localization of nitric oxide synthase I (NOS I) and

Gutiérrez, J.M., Núñez, J., Díaz, C., Cintra, A.C., Homsi-Brandeburgo, M.I. & Giglio, J.R.

Habermann, E., Dreyer, F. & Bigalke, H. (1980). Tetanus toxin blocks the neuromuscular

*Pharmacology*, Vol.311, No.1, (February 1980), pp. 33-40, ISSN 0028-1298 Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B & Lalloo, D.G (2009). Snake

*Research*, Vol.1, No.7, (September 2010), pp. 1-7, ISSN 2444-0324

*vivo* animal models. *The Journal of Nutrition,* Vol.134, No.12, (December 2004), pp.

bioavailability and effects on health. *Natural Product Reports,* Vol.26, No.8, (May

extract of black tea (*Camellia sinensis*) on the rat diaphragm. *Planta Medica,* Vol.60,

(*Camellia sinensis*) extract on the rat diaphragm. *Journal of Ethnopharmacology*, Vol.57,

Oshima-Franco Y. (2010). The inhibitory effect of *Camellia sinensis* extracts against the neuromuscular blockade of *Crotalus durissus terrificus* venom. *Journal of Venom*

A.C., Braz, A.S., Soares, A.M. & Fontes, M.R. (2011). Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase

molecular evidence for phylogeny and classification of South American pitvipers, genera *Bothrops*, *Bothriopsis*, and *Bothrocophias* (Serpentes: Viperidae). *Zoological Journal of the Linnean Society*, Vol.156, No.3, (March 2009), pp. 617-640, ISSN 0024-4082

of tea flavonoids and teas. *Molecular Nutrition & Food Research*, Vol.51, No.1,

of snake venom Lys49 phospholipase A2 homologues. *Toxicon,* Vol.55, No.10,

NMDA receptor subunit 1 (NMDAR-1) at the neuromuscular junction in rat and mouse skeletal muscle. *Cell and Tissue Research*. Vol.291, No.1, (January 1998),

(1991). Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake *Bothrops jararacussu*. *Experimental and Molecular Pathology,* Vol.55, No.3, (December 1991), pp.

transmission *in vitro* like botulinum A toxin. *Naunyn-Schmiedeberg's Archives of*

envenoming: a disease of poverty. *PLoS Neglected Tropical Diseases*, Vol.3, No.12,

#### **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.

#### **7. References**

*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

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

Arab, L. & Il'yasova, D. (2003). The epidemiology of tea consumption and colorectal cancer

Basu, S., Chaudhuri, T., Chauhan, S.P.S., Das Gupta, A.K., Chaudhury, L. & Vedasiromoni,

Battellino, C., Piazza, R, da Silva, A.M., Cury, Y. & Farsky, S.H. (2003). Assessment of efficacy

Bülbring, E. (1946). Observation on the isolated phrenic nerve diaphragm preparation of the

Chippaux, J.P. (2011). Estimate of the burden of snakebites in sub-Saharan Africa: a meta-

Cintra, A.C., Marangoni, S., Oliveira, B. & Giglio, J.R. (1993). Bothropstoxin-I: amino acid

Cintra-Francischinelli, M., Silva, M.G., Andréo-Filho, N., Gerenutti, M., Cintra, A.C.,

Cintra-Francischinelli, M., Pizzo, P., Rodrigues-Simioni, L., Ponce-Soto, L.A., Rossetto, O.,

Correa-Netto, C., Teixeira-Araujo, R., Aguiar A.S., Melgarejo, A.R., De-Simone, S.G., Soares,

toxins. *Toxicon,* Vol.55, No.7, (June 2010), pp. 1222-1235, ISSN 0041-0101

incidence. *The Journal of Nutrition* Vol 133, No. 10, (October 2003), pp. 3310S–3318S,

J.R. (2005). The theaflavin fraction is responsible for the facilitatory effect of black tea at the skeletal myoneural junction. *Life Sciences*, Vol.76, No.26, (May 2005), pp.

of bothropic antivenom therapy on microcirculatory effects induced by *Bothrops jararaca* snake venom. *Toxicon*, Vol.41, No.5, (April 2003), pp. 583-593, ISSN 0041-0101

rat. *British Journal of Pharmacology and Chemotherapy,* Vol.1, (May 1946), pp. 38-61,

analytic approach. *Toxicon,* Vol.57, No.4, (March 2011), pp. 586-599. ISSN 0041-0101

sequence and function. *Journal of Protein Chemistry*, Vol.12, No.1, (February 1993),

Giglio, J.R., Leite, G.B., Cruz-Höfling, M.A., Rodrigues-Simioni, L. & Oshima-Franco, Y. (2008). Antibothropic action of *Casearia sylvestris* Sw. (Flacourtiaceae) extracts. *Phytotherapy Research*, Vol.22, No.6, (June 2008), pp. 784-790, ISSN 0951-418X

Lomonte, B., Gutiérrez, J.M., Pozzan, T. & Montecucco, C. (2009). Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors. *Cellular and Molecular Life Science*, Vol.66, No.10, (March 2009), pp.

M.R., Foguel, D. & Zingali, R.B. (2010). Immunome and venome of *Bothrops jararacussu*: a proteomic approach to study the molecular immunology of snake

gallate have a strong participation on these protective effects.

**5. Conclusion**

**6. Acknowledgment**

**7. References**

scholarship (I.C.) from PROBIC/UNISO.

3081–3088, ISSN 1879-0631

pp. 57-64, ISSN 0277-8033

1718–1728, ISSN 1420-682X

ISSN 1541-6100

ISSN 0366-0826

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

<http://dtr2004.saude.gov.br/sinanweb/index.php>

Vol.11, No.4, (April 2000), pp. 216–222, ISSN 0955-2863

661, ISSN 0037-8682

pp. 175–182, ISSN 1742-7835

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 585

Miura, Y., Chiba, T., Miura, S., Tomita, I., Umegaki, K., Ikeda, M. & Tomita, T. (2000). Green

Nachshen, D.A. (1984). Selectivity of the Ca binding site in synaptosome Ca channels:

Nicoleti, A.F., de Medeiros, C.R., Duarte, M.R. & de Siqueira França, F.O. (2010).

Oshima-Franco, Y., Leite, G.B., Dal Belo, C.A., Hyslop, S., Prado-Franceschi, J., Cintra,

Pereira, M.F., Novello, J.C., Cintra, A.C., Giglio, J.R., Landucci, E.T., Oliveira, B. & Marangoni,

Rodrigues-Simioni, L., Borgese, N. & Ceccarelli, B. (1983). The effects of *Bothrops jararacussu*

Rubem-Mauro, L., Rocha Jr, D.S., Barcelos, C.C., Costa-Varca, G.H., Andréo-Filho, N.,

*Pharmacy, Vol.* 28, No.2, (February 2009), pp. 211-218, ISSN 0326-2383 Satoh, E., Ishii, T., Shimizu, Y., Sawamura, S. & Nishimura, M. (2002a). The mechanism

*Protein Chemistry*, Vol.17, No.4, (May 1998), pp. 381-386, ISSN 0277-8033 Pithayanukul, P., Leanpolchareanchai, J. & Bavovada, R. (2010). Inhibitory effect of tea

*Research*, Vol.24, No.S1, (July 2009), pp. S56–S62, ISSN 0951-418X

No.2, (October 1983), pp. 475-489, ISSN 0306-4522

2002), pp. 441-447, ISSN 0388-1350

*Physiology*, Vol.83, No.6, (June 1984), pp. 941–967, ISSN0022-1295

clinicopathological studies of 29 proven cases in São Paulo State, Brazil. *The Quarterly Journal of Medicine,* Vol.90, No.5, (May 1997), pp. 323-334, ISSN 0033-5622 Ministério da Saúde. (2004). Secretaria de Vigilância em Saúde. Sistema de Informação de

Agravos de Notificação (SINAN), 07.05.2010, Available from:

tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an *ex vivo* study in humans. *The Journal of Nutricional Biochemistry*,

inhibition of Ca influx by multivalent metal cations. *The Journal of General*

Comparison of *Bothropoides jararaca* bites with and without envenoming treated at the Vital Brazil Hospital of the Butantan Institute, State of São Paulo, Brazil. *Revista da Sociedade Brasileira de Medicina Tropical*, Vol.43, No.6, (December 2010), pp. 657-

A.C.O., Giglio, J.R., Cruz-Höfling, M.A., & Rodrigues-Simioni, L. (2004). The presynaptic pctivity of bothropstoxin-I, a myotoxin from *Bothrops jararacussu* snake venom. *Basic & Clinical Pharmacology & Toxicology*, Vol. 95, No. 4, (October 2004),

S. (1998). The amino acid sequence of bothropstoxin-II, an Asp-49 myotoxin from *Bothrops jararacussu* (Jararacuçu) venom with low phospholipase A2 activity. *Journal of*

polyphenols on local tissue damage induced by snake venoms. *Phytotherapy*

venom and its components on frog nerve-muscle preparation. *Neuroscience*, Vol.10,

Barberato-Filho, S., Oshima-Franco, Y. & Vila, M.M.D.C. Phenobarbital pharmacological findings on the nerve-muscle basis. *Latin American Journal of*

underlying the protective effect of the thearubigin fraction of black tea (*Camellia sinensis*) extract against the neuromuscular blocking action of botulinum neurotoxins. *Pharmacology & Toxicology*, Vol.90, No.4, (April 2002), pp. 199-202, ISSN 0901-9928 Satoh, E., Ishii, T., Shimizu, Y., Sawamura, S. & Nishimura, M. (2002b). A mechanism of the

thearubigin fraction of black tea (*Camellia sinensis*) extract protecting against the effect of tetanus toxin. *The Journal of Toxicological Sciences*, Vol.27, No.5, (December

characterization of melanic pigments derived from tea and tea polyphenols. *Food*

(2004). In: *Farmacognosia: da Planta ao Medicamento*, C.M.O. Simões, E.P. Schenkel,

Sava, V., Yang, S-M., Hong, M-Y., Yan, P-C. & Huang, G.S. (2001). Isolation and

*Chemistry*, Vol.73, No.2, (March-April 2001), pp. 177–184, ISSN 0308-8146 Simões, C.M.O., Schenkel, E.P., Gosmann, G., Mello, J.C.P., Mentz, L.A. & Petrovick, P.R.

Hazarika, M., Chakravarty, S. K. & Mahanta, P. K. (1984). Studies on thearubigin pigments

Heluany, N.F., Homsi-Brandeburgo, M.I., Giglio, J.R., Prado-Franceschi, J. & Rodrigues-

Homsi-Brandeburgo, M.I., Queiroz, L.S., Santo-Neto, H., Rodrigues-Simioni, L. & Giglio, J.R.

Hughes, B.W., Kusner, L.L. & Kaminski, H.J. (2006). Molecular architecture of the

Hung, Y-C., Sava, V., Hong, M-Y. & Huang, G.S. (2004). Inhibitory effects on phospholipase

Khan, N. & Mukhtar, H. (2007). Tea polyphenols for health promotion. *Life Sciences*, Vol.81,

Krishnamoorthy, K.K. (1991). The nutritional and therapeutic value of tea. In: Yamanishi, T.

Lambert, J.D. & Yang, C.S. (2003). Mechanisms of cancer prevention by tea constituents. *The*

Leung, L.K., Su, Y., Chen, R., Zhang, Z., Huang, Y. & Chen, Z-Y. (2001). Theaflavins in

*Nutrition*, Vol.131, No.9, (September 2001), pp. 2248-2251, ISSN 0022-3166 Lima, A.C.S.F., Campos, C.E.C. & Ribeiro, J.R. (2009). Epidemiological profile of snake

action. *Toxicon*, Vol. 42, No. 8, (December 2003), pp. 885–901, ISSN 0041-0101 Mahmood, A., Ahmad, M., Jabeen, A., Zafar, M. & Nadeem, S. (2005). *Pharmacognostic*

Malomouzh, A.I., Mukhtarov, M.R., Nikolsky, E.E. & Vyskocil, F. (2007). Muscarinic M1

Melo, R.F., Farrapo, N.M., Rocha Junior, D.S., Silva, M.G., Cogo, J.C., Dal Belo, C.A.,

Milani Júnior, R., Jorge, M.T., de Campos, F.P., Martins, F.P., Bousso, A., Cardoso, J.L.,

Vol.102, No.6, (September 2007), pp. 2110-2117, ISSN 0022-3042

*Tropical*, Vol.42, No.3, (May-June 2009), pp. 329-335, ISSN 0037-8682 Lomonte, B., Angulo, Y. & Calderón, L. (2003). An overview of lysine-49 phospholipase A2

Vol.35, No.11, (November 1984), pp. 1208-1218, ISSN 0022-5142

No.10, (October 1992), pp. 1203-1210, ISSN 0041-0101

Vol.74, No.16, (March 2004), pp. 2037-2047, ISSN 1879-0631

(July 1988), pp. 615-627, ISSN 0041-0101

No.7, (July 2007), pp. 519-533, ISSN 1879-0631

http://www.siu.edu/~ebl/leaflets/abid.htm

60741-622-7, New York, United States of America

ISSN 0148-639X

Control Number 92188482

in black tea manufacturing systems. *Journal of the Science of Food and Agriculture,*

Simioni, L. (1992). Effects induced by bothropstoxin, a component from *Bothrops jararacussu* snake venom, on mouse and chick muscle preparations. *Toxicon*, Vol.30,

(1988). Fractionation of *Bothrops jararacussu* snake venom: partial chemical characterization and biological activity of bothropstoxin. *Toxicon*, Vol.26, No.7,

neuromuscular junction. *Muscle & Nerve,* Vol. 33, No.4, (October 2005), pp. 445–461,

A2 and antivenin activity of melanin extracted from *Thea sinensis* Linn. *Life Sciences*,

(Ed.), *Proceedings of the International Symposium on Tea Science*, Japan, pp. 6–11, LC

*Journal of Nutrition,* Vol.133, No.10, (October 2003), pp. 3262S-3267S, ISSN 0022-3166

black tea and catechins in green tea are equally effective antioxidants. *The Journal of*

poisoning accidents in the State of Amapá. *Revista da Sociedade Brasileira de Medicina*

myotoxins from crotalid snake venoms and their structural determinants of myotoxic

*studies of some indigenous medicinal plants of Pakistan*, 26.07.2011, Available from:

acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism. *Journal of Neurochemistry*,

Rodrigues-Simioni, L., Groppo, F.C. & Oshima-Franco, Y. (2009). Antiophidian mechanisms of medicinal plants, In: *Flavonoids: Biosynthesis, Biological Effects and Dietary Sources,* R. B. Keller. (Ed.), 249-262, Nova Science Publishers, ISBN 978-1-

Ribeiro, L.A., Fan, H.W., França, F.O., Sano-Martins, I.S., Cardoso, D., Ide Fernandez, C., Fernandes, J.C., Aldred, V.L., Sandoval, M.P., Puorto, G., Theakston, R.D. & Warrell, D.A. (1997). Snake bites by the jararacuçu (*Bothrops jararacussu*): clinicopathological studies of 29 proven cases in São Paulo State, Brazil. *The Quarterly Journal of Medicine,* Vol.90, No.5, (May 1997), pp. 323-334, ISSN 0033-5622

**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/>.


Game Theory - From Idea to Practice

*Edited by Branislav Sobota*

Published in London, United Kingdom

Game Theory - From Idea to Practice http://dx.doi.org/10.5772/intechopen.102220 Edited by Branislav Sobota

#### Contributors

Ian M. Robinson, Mark Flanagan, Trevor C. Lipscombe, Adrian Northey, Matheus Cezarotto, Pamela Martinez, Barbara Chamberlin, Javier Vilamitjana, Julio Calleja-Gonzalez, Diego Marqués-Jiménez, Klaus P. P. Jantke, Hans-Holger Wache, Ronny Franke, David Ginat, Yasuo Nakayama, Corey Reutlinger, Branislav Sobota, Emília Pietriková, Merav Hayak, Orit Avidov-Ungar

#### © The Editor(s) and the Author(s) 2023

The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department (permissions@intechopen.com).

Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be found at http://www.intechopen.com/copyright-policy.html.

#### Notice

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2023 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, 5 Princes Gate Court, London, SW7 2QJ, United Kingdom

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library

Additional hard and PDF copies can be obtained from<EMAIL_ADDRESS>
Game Theory - From Idea to Practice Edited by Branislav Sobota p. cm. Print ISBN 978-1-83768-059-7 Online ISBN 978-1-83768-060-3 eBook (PDF) ISBN 978-1-83768-061-0