**4.2.** *In vitro* **myotoxic activity assay**

20 Chromatography – The Most Versatile Method of Chemical Analysis

(CK-MM), cardiac (CK-MB) and in the brain (CK-BB).

two main enzymes used to this end:

isolated from snake venom [23].

periphery blood or to the supernatant of the culture medium of cellular lineages. There are

Creatine Kinase (EC 2.7.3.2): is a dimeric protein formed by the combination of subunits (B and M) and in its cytosolic form is found in many tissues, especially in skeletal muscle tissue

Lactate dehydrogenase (EC 1.1.1.27): is an enzyme widely distributed in many tissues and organisms. It is presented in the form of homo or hetero tetramers of subunits M and H,

*In vivo*, the CK activity quantification in murine models has been the most used to assay the presence of myotoxic PLA2, especially due to their low cost, ease of performance and high

As for the *In vitro* assays, myoblast lineages C2C12 (ATCC CRL-1772), differentiated until the formation of myotubules, have been used as models to assay the cellular toxicity, through the

Regarding the phospholipase activity detections, it can be done by direct and indirect methods. Directly, it is possible to detect the presence of PLA2s with the use of chromogenic substrates, such as 4N3OBA(4-nitro-3-octanoyloxybenzoic acid) that induce the formation of detectable product at 425 nm [65] and fluorescent substrates (NBD) coupled to phospholipids that are used to quantitively and qualitatively survey the PLA2s activity

Indirectly, the approach used consists in the potentiometric assay of the fatty acids released after the enzymatic hydrolysis of the phospholipids, through the quantification with standard alkaline solution [66]. Moreover, fatty acids released by the enzymatic degradation can be quantified through the alteration of the optical density of the pH indicator solution, such as phenol red [67], brilliant yellow [68] and bromothymol blue [69]. Another indirect method to assay PLA2 activity present in samples consists in the detection of hemolysis induced by lysophospholipids derived from phospholipids submitted to enzymatic digestion. This can be done through the quantification of hemoglobin present in solution or through the visualization

Mice is used for the *in vivo* assay of the myotoxic activity. Swiss males weighing between 18 g and 22 g, kept in controlled environment (12 h in the light and 12 h in the dark), with food and water *ad libitum* up to the moment of use. PBS solubilized sample and control (PBS) are filtered through 44 µm pores immediately prior to use. Reagents for CK activity dosage are

A Sample (50 µL) or control (50 µL) will be injected in mice gastrocnemic muscle using adequate device in order to guarantee a precise volume control. After a time lap (3 and/or 6 h), blood sample is collected in heparinized tubes and centrifuged to separate plasma. CK

being present in muscular tissue in the homotetrameric form of subunit M.

specificity as skeletal muscular tissue lesion markers when exposed to myotoxins.

quantification of LDH levels in the supernatant of cell cultures exposed to toxins.

of hemolytic halo in agarose matrix with immobilized red blood cells.

prepared and used according to manufacturer's instructions.

**4.1.** *In vivo* **assay of the myotoxic activity** 

In order to assay myotoxic *in vitro* activity, myoblast lineage cells are used, such as murine skeletal muscle C2C12 myoblasts (ATCC CRL-1772) as described by Lomonte et al. [73], cultivated in modified Dubelco Eagle medium, supplemented with 1% bovine fetal serum. PBS solubilized sample, negative control (PBS) and positive control (Triton X-100) should be filtered through 22 µm pore filters immediately prior to use. Reagents for LDH activity dosage are prepared and used according to manufacturer's instructions.

In 96 well plate, 2X105 cells/150 µL are set, sample and/or control (50 µL) are incubated in humid atmosphere at 37 °C and 5% CO2 for a 3 hour period. Afterwards, collect supernatant aliquot and quantify LDH activity released by cells with cytoplasmic membrane integrity compromised, according to manufacturer's instructions and expressed in U/I, where one unit corresponds to the production of 1 mmol of lactate per minute.
