**1. Introduction**

568 Pharmacology

Wen T, Wu ZM, Liu Y, Tan YF, Ren F, and Wu H (2007) Upregulation of heme oxygenase-1

White RE and Coon MJ (1980) Oxygen activation by cytochrome P-450. *Annu.Rev Biochem.*

Williams SE, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, Peers C, and Kemp PJ

Wright MM, Schopfer FJ, Vidyasagar V, Powell P, Chumley P, Iles KE, Freeman BA, and

Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, and

Yang L, Quan S, and Abraham NG (1999) Retrovirus-mediated HO gene transfer into

Yao P, Hao L, Nussler N, Lehmann A, Song F, Zhao J, Neuhaus P, Liu L, and Nussler A

Yao P, Nussler A, Liu L, Hao L, Song F, Schirmeier A, and Nussler N (2007) Quercetin

Yoshioka K, Deng T, Cavigelli M, and Karin M (1995) Antitumor promotion by phenolic

Young SC, Storm MV, Speed JS, Kelsen S, Tiller CV, Vera T, Drummond HA, and Stec DE

Zangar RC, Davydov DR, and Verma S (2004) Mechanisms that regulate production of reactive oxygen species by cytochrome P450. *Toxicol.Appl.Pharmacol.* 199:316-331. Zhang J, Ohta T, Maruyama A, Hosoya T, Nishikawa K, Maher JM, Shibahara S, Itoh K, and

induction in response to oxidative stress. *Mol.Cell Biol.* 26:7942-7952.

heme oxygenase-1 via the MAPK/Nrf2 pathways. *J.Hepatol.* 47:253-261. Yoneya R, Ozasa H, Nagashima Y, Koike Y, Teraoka H, Hagiwara K, and Horikawa S (2000)

heme oxygenase-1 deficiency. *J.Clin.Invest* 103:129-135.

chloride in the rat. *Toxicol.Lett.* 116:223-229.

*Proc.Natl.Acad.Sci.USA* 92:4972-4976.

hepatic injury in rats. *Toxicology* 237:184-193.

channel. *Science* 306:2093-2097.

49:315-356.

103:4299-4304.

*Physiol.* 296:G1318-1323.

297:R1546-R1553.

with hemin prevents D-galactosamine and lipopolysaccharide-induced acute

(2004) Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium

Agarwal A (2006) Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid potently activates endothelial heme oxygenase 1 expression. *Proc.Natl.Acad.Sci.*

Koizumi S (1999) Oxidative stress causes enhanced endothelial cell injury in human

endothelial cells protects against oxidant-induced injury. *Am.J.Physiol.* 277:127-133.

(2009) The protective role of HO-1 and its generated products (CO, bilirubin, and Fe) in ethanol-induced human hepatocyte damage. *Am.J.Physiol.Gastrointest.Liver* 

protects human hepatocytes from ethanol-derived oxidative stress by inducing

Hemin pretreatment ameliorates aspects of the nephropathy induced by mercuric

antioxidants: inhibition of AP-1 activity through induction of Fra expression.

(2009) Inhibition of biliverdin reductase increases ANG II-dependent superoxide levels in cultured renal tubular epithelial cells. *Am.J.Physiol.Integr.Comp.Physiol.*

Yamamoto M (2006) BRG1 interacts with Nrf2 to selectively mediate HO-1

Snake bite envenoming, a serious public health problem in rural areas of tropical and subtropical countries, was included in 2007 as a neglected disease by the World Health Organization (WHO, 2007). Under this geographical perspective Africa, Asia, Oceania and Latin America are the most vulnerable countries to this kind of accident, but also shared by many developing countries (Harrison et al*.*, 2009; Warrel, 2010). An excellent meta-analytic approach about the subject was described by Chippaux (2011), who analysed more than 3,000 references for estimating the burden of snakebites in sub-Saharan Africa. Brazil encloses both requirements, as a developing and a tropical country, and needs to strengthen measures against venomous snake accidents, since, according to Lima et al*.* (2009), it is the country with the major number of accidents (about 20,000 cases/year), followed by Peru (4,500), Venezuela (2,500-3,000), Colombia (2,675), Ecuador (1,200-1,400) and Argentina (1,150-1.250) (Warrel, 2004).

As mentioned by Nicoleti et al*.* (2010), venomous snakes in Brazil are represented by *Bothrops, Bothropoides, Bothriopsis, Bothrocophias, Rhinocerophis, Crotalus*, *Lachesis, Leptomicrurus* and *Micrurus* (see the new taxonomic arrangement proposed by Fenwick et al*.*, 2009). Envenoming by the first five genera produce similar toxic manifestations and treatment assessment are quite the same. They represent 86.9% of accidents, whereas 8.7% were caused by *Crotalus*, 3.6% *Lachesis* and 0.8% by *Leptomicrurus* and *Micrurus* (Ministério da Saúde, 2004).

*Bothrops jararacussu* snake belongs to the Viperidae family and its venom is able to induce severe signs of local and systemic envenoming, such as necrosis, shock, spontaneous

<sup>\*</sup> Luana de Jesus Reis Rosa1, Gleidy Ana Araujo Silva1, Jorge Amaral Filho1, Magali Glauzer Silva1, Patricia Santos Lopes2, José Carlos Cogo3, Adélia Cristina Oliveira Cintra4 and Maria Alice da Cruz-Höfling5

<sup>1</sup>*University of Sorocaba/UNISO, Brazil*

<sup>2</sup>*Federal University of São Paulo/UNIFESP, Brazil* 

*<sup>3</sup>University of Vale do Paraiba/UNIVAP, Brazil* 

*<sup>4</sup>University of São Paulo/USP, Brazil* 

*<sup>5</sup>University of Campinas/UNICAMP/I.B./D.H.E., Brazil*

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

**2.1 Hydroalcoholic extract from leaves of** *Camellia sinensis*

*sinensis* extract, were also tested.

**2. Materials and methods** 

at room temperature until the assays.

Brazilian College for Animal Experimentation.

**2.2 Pharmacological study** 

**2.2.2 Venom and toxin** 

**2.2.1 Animals** 

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 571

The literature describing the medicinal benefits of tea is extensive, but the report about its consumption to alleviate post game fatigue in players and sportsmen (Krishnamoorthy, 1991) inspired further studies on the mammalian skeletomotor apparatus (Das et al*.*, 1994; 1997). For example, Basu et al*.* (2005) attributed to theaflavin, but not thearubigin, the facilitatory effect induced at the skeletal myoneural junction. This experimental model has been traditionally used for the pharmacological characterization of snake venoms, and the association between *C. sinensis* and snake venoms was a natural consequence. Thus, results showing the inhibitory effect of tea polyphenols on local tissue damage induced by snake venoms (Pithayanukul et al*.*, 2010), and the inhibitory effect of *Camellia sinensis* leaves extracts against the neuromuscular blockade of *Crotalus durissus terrificus* venom (de Jesus Reis Rosa et al*.*, 2010) were recently published. Here, using the same experimental procedure, the antivenom property of *Camellia sinensis* leaves extract was assayed against *Bothrops jararacussu* venom and its main myotoxin, bothropstoxin-I. Commercial theaflavin (from black tea) and epigallocatechin gallate (from green tea), known to be part of the *C.* 

The leaves of *C. sinensis* were harvested from plants growing in an orchard at the University of Sorocaba – UNISO (Sorocaba, SP, Brazil). A voucher specimen was deposited in the Instituto Agronômico de Campinas (IAC, number 50.469) herbarium (http://herbario.iac.sp.gov.br) after identification by L.C. Bernacci. Briefly, sixty-four grams of leaves powder were macerated along with 150 mL of 70° GL ethanol, over 3 days. After this period, the resulting suspension was placed into a percolator with 50 mL of 70° GL ethanol, and left for a further 3 days. The macerated drug was percolated and a 20% hydroalcoholic extract was obtained (de Jesus Reis Rosa et al*.*, 2010). The solvent was evaporated until dryness, and the dried extract was then protected from light and humidity

Male Swiss white mice (26-32 g) were supplied by the Anilab - Animais de Laboratório (Paulínia, São Paulo, Brazil). The animals were housed at 25 ± 3°C on a 12-h light/dark cycle with access to food and water *ad libitum*. This study was approved (protocol number A077/CEP2007) by the Committee for Ethics in Research from the University of Vale do Paraiba (UNIVAP) and all experiments were performed according to the guidelines of the

The crude venom was obtained from adult *Bothrops jararacussu* (Bjssu) snakes (Serpentário do Centro de Estudos da Natureza) and certified by Prof. Dr. Jose Carlos Cogo, University of Vale do Paraiba (Univap), São Jose dos Campos, SP, Brazil. Bothropstoxin-I (BthTX-I) was

obtained under the conditions described by Homsi-Brandeburgo et al*.* (1988).

systemic bleeding and renal failure, incoagulable blood and death (Milani et al*.*, 1997); its venom also blocks *in vitro* the contractile skeletal muscle response (Rodrigues-Simioni et al*.*, 1983). Two myotoxins are responsible for myonecrosis: bothropstoxin-I (Homsi-Brandeburgo et al*.*, 1988), the first myotoxin isolated from the venom that reproduces the effects of the crude venom (Heluany et al*.*, 1992), further characterized as a phospholipase A2-Lys49 (Cintra et al*.*, 1993); and bothropstoxin-II, a phospholipase A2 (Gutiérrez et al*.*, 1991), further characterized as an Asp49-PLA2 myotoxin with low catalytic activity (Pereira et al*.*, 1998), although phylogenetically it is more related to Lys49-PLA2s than to other Asp49-PLA2s (dos Santos et al*.*, 2011).

Snake antivenom immunoglobulins (antivenoms) are the only specific treatment for envenoming by snakebites. They are produced by fractionation of plasma usually obtained from large domestic animals hyper-immunized against relevant snake venoms. When injected into an envenomed human patient, antivenom will neutralize any of the effects of the venoms used in its production, and in some instances will also neutralize effects of venoms from closely related species (WHO, 2011a). However, the antibothropic serum effectiveness against the local effects of *Bothrops jararacussu* venom (one of the bothropic venoms used in the serum production) has been debated since the 80's decade (see Correa-Neto et al., 2010). A possible explanation for the lack of effectivenes was given by Battellino et al. (2003) through the use of intravital microscopy after intravenous administration of antibothropic antivenom (BAv), labeled with fluorescein isothiocyanate (FITC). They observed that the antivenom neutralized the systemic effects, but did not efficiently reverse the local effects due to an impaired and/or delayed venom:antivenom interaction at the site of injury. Considering that local effect of venomous snakebites are poorly prevented by specific antivenom, that the access to public health services by people of distant rural regions in tropical and subtropical countries is in general difficult, the use of medicinal plants as a local solution has been a practice of natives of those regions.

Medicinal plants represent a sophisticated biotechnological laboratory that is able to produce a multitude of pharmacologically bioactive substances, with a wide variety of effects (Mahmood et al., 2005). The second beverage (next to water) of major consumption in the world, in its green, black and oolong forms, is the tea from *Camellia sinensis* L. leaves Compounds as polyphenols, polysaccharides, aminoacids, vitamins (Crespy & Williamson, 2004), caffeine and a very small amout of methylxanthines (Yang et al., 1998) can be found in the plant. Catechins, the major component of green tea (fresh leaves are steamed to prevent fermentation, yielding a dry, stable product), and represent the low-molecular-weight polyphenols consisting mainly of flavanol (flavan-3-ol) monomers, such as epicatechin, epicatechin-3-gallate, epigallocatechin and the major, 50-80% of the total catechin, epigallocatechin-3-gallate (Graham, 1992; Khan & Mukhtar, 2007). Catechins account for 6- 16% (Zhu & Chen, 1999) up to 30-40% (Phithayanukul et al*.*, 2010) of the dry green tea leaves. The fermentation or semifermentation stage (when the withered leaves are rolled and crushed) during the manufacture of black or oolong tea, respectively, converts catechins to theaflavins (theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3' digallate, accounting for 3-6% of solid extract) and thearubigins (accounting for 12-18% of solid extract) (Leung et al*.*, 2001; Khan & Mukhtar, 2007), which are complex polyphenols of poorly-defined chemical structures formed during fermentation of polymerization of theaflavins (Hazarika et al*.*, 1984).

The literature describing the medicinal benefits of tea is extensive, but the report about its consumption to alleviate post game fatigue in players and sportsmen (Krishnamoorthy, 1991) inspired further studies on the mammalian skeletomotor apparatus (Das et al*.*, 1994; 1997). For example, Basu et al*.* (2005) attributed to theaflavin, but not thearubigin, the facilitatory effect induced at the skeletal myoneural junction. This experimental model has been traditionally used for the pharmacological characterization of snake venoms, and the association between *C. sinensis* and snake venoms was a natural consequence. Thus, results showing the inhibitory effect of tea polyphenols on local tissue damage induced by snake venoms (Pithayanukul et al*.*, 2010), and the inhibitory effect of *Camellia sinensis* leaves extracts against the neuromuscular blockade of *Crotalus durissus terrificus* venom (de Jesus Reis Rosa et al*.*, 2010) were recently published. Here, using the same experimental procedure, the antivenom property of *Camellia sinensis* leaves extract was assayed against *Bothrops jararacussu* venom and its main myotoxin, bothropstoxin-I. Commercial theaflavin (from black tea) and epigallocatechin gallate (from green tea), known to be part of the *C. sinensis* extract, were also tested.
