**2. Materials and methods**

570 Pharmacology

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

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

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

plants as a local solution has been a practice of natives of those regions.

Asp49-PLA2s (dos Santos et al*.*, 2011).

theaflavins (Hazarika et al*.*, 1984).

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

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 at room temperature until the assays.

#### **2.2 Pharmacological study**

#### **2.2.1 Animals**

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 Brazilian College for Animal Experimentation.

#### **2.2.2 Venom and toxin**

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).

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

**2.5 Statistical analysis** 

**3.1 Pharmacological assays 3.1.1 BthTX-I neutralization** 

0

20

40

60

Twitch tension (%)

80

100

120

compared with the basal response of PND.

\*

\*

bothropstoxin-I (BthTX-I); W, washing.

\*

Tyrode control (n=7)

BthTX-I (20 µg/mL, n=11)

*Camellia sinensis* (0.05 mg/mL, n=7)

**3. Results** 

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 573

Each pharmacological protocol was repeated at least three times. Results were expressed as the mean ± standard error of the mean (SEM). The Student's *t*-test or repeated measures ANOVA

Figure 1 shows the PND blockade activity of BthTX-I (20 µg/mL, n=11), which was irreversible even after washing (W) of preparations with fresh nutritive Tyrode solution. However, the previous incubation of the toxin with 0.05 mg/mL *Camellia sinensis* extract totally (100%) prevented the characteristic neurotransmission blockade, showing a better functional outcome of neuromuscular preparation after washing. The 0.05 mg/mL of *Camellia sinensis* extract was chosen in all protocols since it induced minor changes

*Camellia sinensis* (0.05 mg/mL) + BthTX-I (20 µg/mL) (n=5)

0 20 40 60 80 100 120 140

Time (min)

Fig. 1. Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Note the total efficacy of C. sinensis extract in protecting the neuromuscular blockade induced by

BthTX-I. Each point represents the mean ± SEM. \* = p<0.05 in comparison with the

\*

\* \*

\* \*

W

\*

\*

were used for statistical comparison of the data. The significance level was set at 5%.

\* \* \* \*

#### **2.2.3 Mouse phrenic-nerve diaphragm muscle (PND) preparation**

The PND was obtained from mice anesthetized with halothane and sacrificed by exsanguination. The diaphragm was removed (Bülbring, 1946) and mounted under a tension of 5 g in a 5 mL organ bath containing continuous-aerated Tyrode solution (control) with the following composition: 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 0.49 mM MgCl2, 0.42 mM NaH2PO4, 11.9 mM NaHCO3, and 11.1 mM glucose. After stabilization with 95% O2/5% CO2, the pH was 7.0. The PND myographic recording was performed according to Melo et al. (2009). Briefly, preparations were stimulated indirectly with supramaximal stimuli (4 x threshold, 0.06 Hz, 0.2 ms) delivered from a stimulator (model ESF-15D, Ribeirão Preto, SP, Brazil) to the nerve through bipolar electrodes. Isometric twitch tension was recorded with a force displacement transducer (cat. 7003, Ugo Basile), coupled to a 2-Channel Recorder Gemini physiograph (cat. 7070, Ugo Basile) via a Basic Preamplifier (cat. 7080, Ugo Basile). PND was allowed to stabilize for at least 20 min before addition of the following substances: BthTX-I alone at 20 µg/mL (n=11); Bjssu alone at 40 µg/mL (n=5); 20 µg/mL BthTX-I + 0.05 mg/mL *C. sinensis* extract (n=5); 40 µg/mL Bjssu + 0.05 mg/mL *C. sinensis* extract (n=3); 40 µg/mL Bjssu + 0.025 mg/mL epigallocatechin gallate (n=3, Sigma-Aldrich, SP, Brazil); 40 µg/mL Bjssu + 0.05 mg/mL theaflavin (n=3); and the controls nutritive Tyrode solution (n=7) and 0.05 mg/mL *C. sinensis* extracts (n=7). The plant extract or commercial phytochemicals concentrations were chosen based on the minor changes obtained in comparison with the basal response of PND incubated with Tyrode nutritive solution (control).

#### **2.3 Quantitative histological study**

At least three preparations (n=3) resulting from pharmacological assays were analyzed by quantitative morfometry. Preparations used in the controls, nutritive Tyrode solution and *C. sinensis* hydroalcoholic extract (0.05 mg/mL) were compared to BthTX-I (20 µg/mL), or *C. sinensis* (0.05 mg/mL) + BthTX-I (20 µg/mL) groups, or Bjssu (40 µg/mL), or *C. sinensis* (0.05 mg/mL) + Bjssu venom (40 µg/mL) after fixation in Bouin solution and submission to routinely morphological techniques. Cross-sections (5 µm thick) of diaphragm muscle embedded in paraffin were stained with Hematoxylin-Eosin for microscopy examination. Tissue damage was expressed in percentage (number of damaged muscle cells divided by the total number of cells in three non-overlapping, non-adjacent areas of each preparation) according to Cintra-Francischinelli et al*.* (2008).

#### **2.4 Thin layer chromatography (TLC)**

Aliquots of *C. sinensis* hydroalcoholic extract were spotted onto 0.2 mm thickness silica gel 60F254 on aluminum plates, 20.10 cm, (Merck, Germany) and developed with ethyl acetate:methanol:water (100:13.5:10, v/v) in a pre-saturated chromatographic chamber along with appropriate phytochemical standards (Simões et al*.*, 2004). These standards (theaflavin and epigallocatechin gallate, Sigma-Aldrich® - USA) were solubilized in methanol (1 mg/mL). The separated spots were visualized (under UV light at 360 nm) with NP/PEG as follows: 5% (v/v) ethanolic NP (diphenylboric acid 2-aminoethyl ester, Sigma Chemical Co., St. Louis, MO, USA) followed by 5% (v/v) ethanolic PEG 4000 (polyethylene glycol 4000, Synth Chemical Co., São Paulo, SP, Brazil). The retention factor (Rf) of each standard was compared with spots exhibited by *C. sinensis* extracts.

## **2.5 Statistical analysis**

Each pharmacological protocol was repeated at least three times. Results were expressed as the mean ± standard error of the mean (SEM). The Student's *t*-test or repeated measures ANOVA were used for statistical comparison of the data. The significance level was set at 5%.
