**3. Results**

572 Pharmacology

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

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

basal response of PND incubated with Tyrode nutritive solution (control).

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)

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

**2.3 Quantitative histological study** 

according to Cintra-Francischinelli et al*.* (2008).

compared with spots exhibited by *C. sinensis* extracts.

**2.4 Thin layer chromatography (TLC)**

#### **3.1 Pharmacological assays**

#### **3.1.1 BthTX-I neutralization**

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 compared with the basal response of PND.

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

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 575

Fig. 3. Cross-sections (5 μm thick) of diaphragm embedded in paraffin and stained with Hematoxylin-Eosin. (A) Control-sham diaphragm preparation (15.9 ± 0.8 %). (B)

Neuromuscular preparation exposed to 0.05 mg/mL Camellia sinensis extract (25.3 ± 1.1 %). (C) Muscle incubated with 20 μg/mL BthTX-I (66.6 ± 2.3 %). (E) Muscle incubated with 40 μg/mL Bjssu venom (75.1 ± 1.1 %). The main fibers damage are lettered as follows: myonecrosis (m), edema (e), delta lesion (arrow), sarcolemmal disruption with nuclei (n) dispersion, "ghost" cells (g) visualized by spaces optically empty. Note that area with extensive myonecrosis has a hyaline aspect. Muscles incubated with 0.05 mg/mL Camellia sinensis extract (D and F) shows fibers maintaining its characteristic polygonal profile in despite of a number of them being edematous (e). A slow percentage of them 23.4 ± 1.3 % for BthTX-I (D); 27.8 ± 0.9 % for Bjssu (F)showed myonecrosis (m). Bars = 50 μm.

#### **3.1.2 Bjssu neutralization**

Figure 2 shows the PND blockade activity by Bjssu crude venom. There was no contraction recovery of PND after washing the preparation. *C. sinensis* extract was 78 ± 12 % able to neutralize the venom that, in turn, differently of its myotoxin, contains several constituents.

Fig. 2. Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Note the partial efficacy of *C. sinensis* extract in protecting the neuromuscular blockade induced by Bjssu. Each point represents the mean ± SEM. \* = p<0.05 in comparison with the venom. W, washing. Bjssu, *Bothrops jararacussu* venom.

#### **3.2 Quantitative histological study**

Figure 3 shows neuromuscular preparations exposed either to Tyrode (Fig. 3A) or *C. sinensis* extract (Fig. 3B): the muscle fibers were well-preserved, showing changes not significantly different between each other of 15.9 ± 0.8 % or 25.3 ± 1.1 % damaged fibers, respectively. These changes were related mainly to loss of the typical cell cross-sectional polygonal profile. Differently, BthTX-I (Fig. 3C, 66.6 ± 2.3 %) and venom (Fig. 3E, 75.1 ± 1.1 %) alone clearly showed in transversal sections characteristic signals of myonecrosis (m), edematous cells (e), loss of polygonal profile, sarcolemma disruption, delta lesion (arrow), "ghost" cells (g), and nuclei (n) dispersed in the tissue. These changes were already extensively described in the scientific literature. Panel 3D and 3F show cross-sections of PND muscle fibers after *in vitro* neutralization by *C. sinensis* extract of BthTX-I (23.4 ± 1.3 % of lesioned fibers, p<0.05) and of Bjssu (27.8 ± 0.9 % of lesioned fibers, p<0.05), respectively.

Figure 2 shows the PND blockade activity by Bjssu crude venom. There was no contraction recovery of PND after washing the preparation. *C. sinensis* extract was 78 ± 12 % able to neutralize the venom that, in turn, differently of its myotoxin, contains several constituents.

*Camellia sinensis* (0.05 mg/mL) + Bjssu (40 µg/mL) (n=3)

0 20 40 60 80 100 120 140

Time (min)

Fig. 2. Isolated mouse phrenic nerve-diaphragm preparations under indirect stimuli. Note the partial efficacy of *C. sinensis* extract in protecting the neuromuscular blockade induced by Bjssu. Each point represents the mean ± SEM. \* = p<0.05 in comparison with the venom.

Figure 3 shows neuromuscular preparations exposed either to Tyrode (Fig. 3A) or *C. sinensis* extract (Fig. 3B): the muscle fibers were well-preserved, showing changes not significantly different between each other of 15.9 ± 0.8 % or 25.3 ± 1.1 % damaged fibers, respectively. These changes were related mainly to loss of the typical cell cross-sectional polygonal profile. Differently, BthTX-I (Fig. 3C, 66.6 ± 2.3 %) and venom (Fig. 3E, 75.1 ± 1.1 %) alone clearly showed in transversal sections characteristic signals of myonecrosis (m), edematous cells (e), loss of polygonal profile, sarcolemma disruption, delta lesion (arrow), "ghost" cells (g), and nuclei (n) dispersed in the tissue. These changes were already extensively described in the scientific literature. Panel 3D and 3F show cross-sections of PND muscle fibers after *in vitro* neutralization by *C. sinensis* extract of BthTX-I (23.4 ± 1.3 % of lesioned fibers, p<0.05)

\*

W

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

**3.1.2 Bjssu neutralization** 

0

20

40

60

Twitch tension (%)

\* \*

W, washing. Bjssu, *Bothrops jararacussu* venom.

and of Bjssu (27.8 ± 0.9 % of lesioned fibers, p<0.05), respectively.

**3.2 Quantitative histological study** 

\*

\* \*

Tyrode control (n=7)

Bjssu (40 µg/mL, n=5)

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

80

100

120

Fig. 3. Cross-sections (5 μm thick) of diaphragm embedded in paraffin and stained with Hematoxylin-Eosin. (A) Control-sham diaphragm preparation (15.9 ± 0.8 %). (B) Neuromuscular preparation exposed to 0.05 mg/mL Camellia sinensis extract (25.3 ± 1.1 %). (C) Muscle incubated with 20 μg/mL BthTX-I (66.6 ± 2.3 %). (E) Muscle incubated with 40 μg/mL Bjssu venom (75.1 ± 1.1 %). The main fibers damage are lettered as follows: myonecrosis (m), edema (e), delta lesion (arrow), sarcolemmal disruption with nuclei (n) dispersion, "ghost" cells (g) visualized by spaces optically empty. Note that area with extensive myonecrosis has a hyaline aspect. Muscles incubated with 0.05 mg/mL Camellia sinensis extract (D and F) shows fibers maintaining its characteristic polygonal profile in despite of a number of them being edematous (e). A slow percentage of them 23.4 ± 1.3 % for BthTX-I (D); 27.8 ± 0.9 % for Bjssu (F)showed myonecrosis (m). Bars = 50 μm.

Antibothropic Action of *Camellia sinensis* Extract Against the Neuromuscular

Blockade by *Bothrops jararacussu* Snake Venom and Its Main Toxin, Bothropstoxin-I 577

Fig. 5. Thin Layer Chromatography performed by using ethyl acetate:methanol:water (100:13.5:10) solvent/Developer: NP/PEG. Phytochemical standards: 1 - Epigallocatechin gallate (Rf=0.80); 2 – Cs, Camellia sinensis leaves extract; 3 – Theaflavin (Rf=0.56). Panel A: chromatoplaque exposed to UV light at 360 nm. Panel B: is the same plaque after NP/PEG

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

chromogenic agent pulverization. Cs spots are suggestive of several flavonoids (yellow/orange fluorescence) and phenolic constituents (blue fluorescence), including epigallocatechin gallate and theaflavin, respectively. Comparative Rf values between

phytochemicals and extract are highlighted in the circle. Rf, retention factor.

**4. Discussion** 

(Rodrigues-Simioni et al*.*, 1983).

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

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 reestablished (not shown).

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.

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

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 pulverization.

Fig. 5. Thin Layer Chromatography performed by using ethyl acetate:methanol:water (100:13.5:10) solvent/Developer: NP/PEG. Phytochemical standards: 1 - Epigallocatechin gallate (Rf=0.80); 2 – Cs, Camellia sinensis leaves extract; 3 – Theaflavin (Rf=0.56). Panel A: chromatoplaque exposed to UV light at 360 nm. Panel B: is the same plaque after NP/PEG chromogenic agent pulverization. Cs spots are suggestive of several flavonoids (yellow/orange fluorescence) and phenolic constituents (blue fluorescence), including epigallocatechin gallate and theaflavin, respectively. Comparative Rf values between phytochemicals and extract are highlighted in the circle. Rf, retention factor.
