An Evaluation of 3-Rhamnosylquercetin, a Glycosylated Form of Quercetin, against the Myotoxic and Edematogenic Effects of sPLA 2 from Crotalus durissus terrificus

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) from Crotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure of C. d. terrificus sPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally, in vitro and in vivo assays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in the C. d. terrificus sPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid against C. d. terrificus sPLA2.


Introduction
At present, phospholipase A 2 s (PLA 2 s) (EC 3.1.1.4) can be classified into various groups and subgroups according to a complex molecular taxonomy. Several PLA 2 s have recently been isolated and characterized. One of the most investigated groups of PLA 2 s includes the secretory phospholipase A 2 s (sPLA 2 ), which are primarily found in the venom of several animals. The sPLA 2 s exhibit well-established functions in the digestion of dietary phospholipids, although they also have important functions in the host's defense against bacterial infections, and they are involved in pathological processes such as atherosclerosis and cancer [1,2]. Moreover, mammalian genomes encode several types of sPLA 2 -binding proteins, indicating that sPLA 2 s may have enzyme-independent activities related to their ability to bind to cellular target proteins [3]. Several recent studies have shown that snake venom sPLA 2 s present a mechanism of action that is very similar to that of human sPLA 2 s [4,5], and some secretory phospholipase A 2 s purified from humans can induce 2 BioMed Research International pharmacological events similar to those of snake venom phospholipase A 2 [6].
Thus, there is great interest in using snake venom sPLA 2 s as molecular target model to evaluate and investigate for natural compounds that potentially inhibit the activities of phospholipase A 2 homologous molecules in other organisms [7][8][9]. This approach could be especially useful for developing better comprehension of several inflammatory diseases, considering the role of sPLA 2 s in the acute inflammation process and the fact that their uncontrolled production can contribute to the exacerbation of these pathological processes [10][11][12]. In this regard, the search for new molecules capable of significantly reducing the enzymatic activity of sPLA 2 and decreasing the production of arachidonic acid through this route is very important from a therapeutic standpoint [13,14].
Various natural compounds have the potential to inhibit or negatively modulate the activities of PLA 2 s and other enzymes involved in the cascade of arachidonic acid, consequently presenting a potential method for reducing and controlling the inflammatory process. The compounds known as flavonoids present remarkable anti-inflammatory activity; these molecules can inhibit the enzymatic activity of PLA 2 s and other enzymes involved in the arachidonic acid pathway, and they can reduce the synthesis of some inflammatory intermediates [15][16][17]. The most common natural flavonoid is quercetin (Q), which is generally found in its glycosylated forms as quercitrin (Qn) or rutin (quercetin rutinoside).
Although some studies indicate that Q has a more pronounced effect in downregulating the inflammatory response relative to Qn, other studies highlight a significant antiinflammatory effect from both glycosides (Qn and rutinoside) in experimental colitis models in rats and other biological essays [17,18]. Thus, the main objective of this work is to clarify, from a structural point of view, the effects of quercitrin's anti-inflammatory properties and the influence of its structural properties on the edema and myonecrosis induced by sPLA 2 purified from C. d. terrificus.
In this study, we performed experimental and theoretical procedures including chromatography, circular dichroism, molecular docking, and other in vitro and in vivo biological essays to evaluate the effects of Q and Qn on C. d. terrificus sPLA 2 . The results obtained from these experiments showed that Qn is a more effective inhibitor of important C. d. terrificus sPLA 2 biochemical and pharmacological activities than Q, indicating that the deoxy sugar rhamnose group is most likely involved in the anti-inflammatory and antimyotoxic properties presented by the glycosylated molecular form of quercetin (Q). , and successively partitioned with hexanes (6.55 g), CH 2 Cl 2 (6.69 g), EtOAc (11.83 g) and n-BuOH (18.87 g). Part of the EtOAc phase (8.0 g) was dissolved with hot methanol, resulting in a precipitate (1.70 g) and a soluble fraction (6.11 g). The soluble portion was then subjected to gel filtration on Sephadex LH-20 eluted with MeOH to make 10 fractions (A1-A10). Fraction A5 (108.8 mg) was subjected to HPLC purification to obtain the Qn flavonoid, which was identified on the basis of its UV, ESI-MS, and NMR data in comparison with data reported in the literature [19].

Purification of Phospholipase A 2 .
To purify the C. d. terrificus sPLA 2 , whole venom was first fractionated as previously described by [20]. Dried venom (45 mg) was dissolved in ammonium bicarbonate buffer (0.2 M, pH 8.0) and clarified by centrifugation (4,500 ×g, 1 min). The supernatant was injected into a molecular exclusion HPLC column (Superdex 75,1 × 60 cm, Pharmacia), and the chromatographic run was performed with a flow rate of 0.2 mL/min for the elution of fractions. The absorbance was monitored at 280 nm. The separated crotoxin-like fraction was immediately lyophilized. The lyophilized fraction was then subjected to reverse-phase chromatography using a -Bondapak C18 column (0.39 × 30 cm) with a flow rate of 1 mL/min for fraction elution. The absorbance was monitored at 280 nm. Afterwards, this fraction was eluted by using a nonlinear gradient with buffer A (0.1% of trifluoroacetic acid in Milli-Q water) and buffer B (acetonitrile 66% in buffer A). The final fraction was the C. d. terrificus sPLA 2 , and its purity was evaluated by tricine SDS-PAGE and mass spectrometry on a MALDI-TOF mass spectrometer as previously described by [21]. 2 with Quercetin and Quercitrin. The incubations of C. d. terrificus sPLA 2 with purified quercetin (Q) and quercitrin (Qn) at (mol : mol) were performed according to the procedure described by [21]. Q and Qn were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO never exceeded 1% during incubation. Q or Qn (400 L of 0.1 mM solution) was added to 400 L of a homogenized, purified C. d. terrificus sPLA 2 solution (1 mg/mL). The mixture was then incubated for 90 min at room temperature, and 200 L aliquots were loaded into a preparative reversephase column to separate the treated enzyme (sPLA 2 : Q and sPLA 2 : Qn). Following column equilibration with HPLC buffer A (aqueous 0.1% TFA), the samples were eluted by using a discontinuous gradient of HPLC buffer B (66.6% of acetonitrile in 0.1% TFA) at a constant flow rate of 1.0 mL/ min. The chromatographic run was monitored at 214 nm.

Circular Dichroism Spectroscopy.
The secondary structure can be determined by CD spectroscopy in the "far-UV" spectral region (190-250 nm). At these wavelengths the chromophore is the peptide bond, and the signal arises when it is located in a regular, folded environment. The CD spectrum of a protein in the "near-UV" spectral region (250-350 nm) is sensitive to certain aspects of tertiary structure of proteins. At these wavelengths the chromophores are the aromatic amino acids and disulfide bonds, and the CD signals they produce are sensitive to the overall tertiary structure of the protein.
In this study, we used both assay types to evaluate the secondary structure and monitor shifts in the tertiary structure of native sPLA 2 and sPLA 2 s that were chemically modified by quercitrin. To determine the protein secondary structure, sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn were dissolved in 10 mM sodium phosphate buffer (pH 7.4), and the final protein concentrations were adjusted to 8.7 mM. This protein solution was then subjected to centrifugation at 4,000 ×g for 5 min, and the resulting supernatant was transferred to a 1 mm pathlength quartz cuvette. Circular dichroism spectra within a wavelength range of 185-300 nm were acquired in-house with a J720 spectropolarimeter (Jasco Corp., Japan) by using a bandwidth of 1 nm and a response time of 1 s. Data collection was performed at room temperature with a scanning speed of 100 nm/min. Nine scans were obtained for each sample, and all spectra were corrected by subtracting buffer blanks. The near-UV CD spectrum (>250 nm) of the samples provided information on the tertiary protein structure. The signals obtained in the range of 250-300 nm were caused by the absorption, dipole orientation, and the nature of the surrounding environment around the phenylalanine, tyrosine, cysteine (or S-S disulfide bridges), and tryptophan residues in the protein. In this study, the CD HPLC detector from Jasco Corp., Japan, was used to enable the scanning of sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn peaks.

Molecular Docking.
For quercitrin (Qn) in silico design and docking simulations, the Avogadro v.0.9.4 (http://avogadro.openmolecules.net/) program was used to generate the in silico model and improve its overall structure through a steepest-descent algorithm for energy minimization based on the MMF94 force field. All docking simulations between the Qn model and the C. d. terrificus sPLA 2 crystallographic structure (PDB ID 2QOG) [22] were executed with the GOLD v.5.0.1 (CCDC Software Limited, Cambridge, UK) program [23]. The docking site was defined within a 10Å radius around the His48 residue located at the catalytic site of monomers A and C of the C. d. terrificus sPLA 2 crystallographic structure. Additionally, other cavities on the protein surface were tested to identify other potential docking sites. The N 1 atoms from the catalytic histidine of the C. d. terrificus sPLA 2 crystallographic model were protonated, and the simulations generated approximately 1000 docking solutions to provide a representative population. The remaining docking parameters were defined according to the GOLD v.5.0.1 default settings. The docking solutions between Qn and the C. d. terrificus sPLA 2 structural model were scored and rescored by using the GoldScore fitness function and the number of H-bonds between the protein and the Qn, respectively. The GoldScore fitness is the sum of the proteinligand bond energy, protein-ligand van der Waals (vdw) energy, ligand internal vdw energy, ligand torsional strain energy, and the ligand intramolecular hydrogen bond energy. This sum represents the amount of docked complex, but it can be excessively high in the case of weak bonds. Therefore, the number of H-bonds between the protein and the Qn was also observed when choosing the best docking solutions [23].

Enzymatic
Assay of sPLA 2 . sPLA 2 activity was measured by following the protocols described in [24] for a 96-well plate assay using 4-nitro-3-octanoyloxy-benzoic acid (NABA or NOB, manufactured by BIOMOL, USA) as the substrate. Enzyme activity, which was expressed as the initial velocity of the reaction (Vo), was calculated on the basis of the increase in absorbance after 20 min. All assays were performed by using = 12 and the absorbance at 425 nm was measured by using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). After the addition of sPLA 2 (20 g), the reaction mixture was incubated for 40 min at 37 ∘ C, and the absorbance was read at 10 min intervals. The effect of the substrate concentration on enzyme activity was determined by measuring the absorbance increase after a 20 min incubation in Tris-HCl buffer, pH 8.0, at 37 ∘ C. All assays were performed in triplicate, and the absorbance at 425 nm was measured by using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). The remaining enzymatic assay was conducted as described above. Q and Qn were dissolved in 1% DMSO.

Paw Edema.
A paw edema assay was performed by using the protocol described by [21]. Male Swiss mice (21 g) were anaesthetized by inhaling halothane. Posterior paw edema was induced by a single subplantar injection of sPLA 2 that was previously incubated with samples of native sPLA 2 and sPLA 2 and previously treated with both flavonoids (10 g per paw). The paw volumes were measured immediately before the injection and at selected time intervals thereafter (0, 30, 60, 120, and 240 minutes) by using a hydroplethysmometer (model 7150, Ugo Basile, Italy). All drugs were dissolved in 0.9% sterile saline solution. The results are expressed as the increase in paw volume (mL) calculated by subtracting the initial volume. The area under the time-course curve was also calculated (trapezoidal rule), and the results were expressed as the total edema volume (milliliters per paw).

Evaluation of Myonecrosis.
The liberation of creatine kinase (CK) from damaged muscle cells was determined by recording the enzyme activity in mouse plasma by using the CK-NAc kit (http://www.laborlab.com.br/, Laborlab, Brazil) as described in [25]. Native sPLA 2 and sPLA 2 were previously treated with both Q and Qn. These samples were injected into the left gastrocnemius muscle of male Swiss mice (18-20 g; = 5). The right gastrocnemius muscle was injected with 50 L of 0.5 mg/mL sPLA 2 samples. Control mice received an equal volume of 0.15 M NaCl. After 3 h, the mice were anesthetized, and blood was collected from the abdominal vena cava into tubes containing heparin as an anticoagulant. The plasma was stored at 4 ∘ C for a maximum of 12 h before the assay. The amount of CK was then determined with 40 L of plasma, which was incubated for 3 minutes at 37 ∘ C with 1.0 mL of the reagent according to the kit protocol. The resulting activity was expressed in U/L.

Statistical Analysis.
Results are reported as the means ± SEM of replicated experiments. The significance of differences between means was assessed by an analysis of variance followed by Dunnett's test when several experimental groups were compared to the control group. The confidence limit for significance was 5%.

Purification of Chemically
Treated sPLA 2 and sPLA 2 . Figure 1(a) shows the chromatography profiles of the eluted native sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn. The retention times of native sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn were 32.5, 31.8, and 33.5 minutes, respectively. All sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn samples were lyophilized and stored for future analysis. Figure 1(b) shows the mass spectrometry profile of native sPLA 2 and sPLA 2 : Q, which was the same as that found by [21]; this finding shows that the methods used here were stable, and they generated reliable and accurate data. Furthermore, the analysis result of sPLA 2 mass spectrometry: Qn was 14580.90, so this mass is the product of sPLA 2 incubation with Qn. Thus, Figure 1(b) only shows the results of sPLA 2 : Qn, and the results of the incubation of the product of sPLA 2 : Q were presented in the Figure 1(b). Figure 2 shows the circular dichroism profile of the native sPLA 2 , sPLA 2 : Qn, and sPLA 2 : Q, which were subjected to the same test conditions. The far-UV region (ultraviolet) ranging between 190 and 260 nm was used to reveal important features of its secondary structure. The results are shown in Figure 2(a), indicating that Qn was able to induce some secondary modifications in native sPLA 2 in comparison with quercetin (Q). In addition, Qn was able to induce a significant change in the random coil region of native sPLA 2 . The near-UV CD spectrum (>250 nm) of protein provides information on the tertiary structure. The signals obtained in the 250-300 nm region are caused by absorption, dipole orientation, and the nature of the environment surrounding the phenylalanine, tyrosine, cysteine (or S-S disulfide bridges), and tryptophan amino acids. Figure 2(b) shows the UV CD spectrum of native sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn, and from these results, the previous native sPLA 2 treatment with Qn induced more evident tertiary shifts than native sPLA 2 . model [22], and they were used to analyze the probable preferential orientation of the ligands (Q and Qn) in a complex with the C. d. terrificus sPLA 2 . Based on the docking scores, this computational analysis showed that Qn has a higher affinity for the active site of C. d. terrificus sPLA 2 than Q. The Avogadro v.0.9.4 (http://avogadro.openmolecules.net/) program was used to generate an in silico model of Qn and improve its overall structure through a steepest-descent algorithm for energy minimization based on the MMF94 force field. All docking simulations between the Qn model and the C. d. terrificus sPLA 2 crystallographic structure (PDB ID 2QOG) [22] were performed with the GOLD v.5.0.1 (CCDC Software Limited, Cambridge, UK) program [23]. The docking site was defined by a 10Å radius around the His48 residue, which was located at the catalytic site of the A and C monomers of the C. d. terrificus sPLA 2 crystallographic structure. Additionally, other cavities on the protein surface were also tested to identify other potential docking sites. The N 1 atoms from the catalytic histidine in the C. d. terrificus sPLA 2 crystallographic model were protonated, and the simulations generated approximately 1000 docking solutions to provide a representative population. The remaining docking parameters were defined according to the GOLD v.5.0.1 default settings. The docking solutions between Qn and the C. d. terrificus sPLA 2 structural model were scored and rescored by using the GoldScore fitness function. As shown in Figure 3, the main interactions between the ligand and the protein involve amino acid residues Asp49, His48, and Gly30 and the Ca 2+ ion.

Enzymatic Assays.
All enzymatic assays yield a product that is linear over a short period of time at an initial rate after the beginning of the enzyme activity (when performed under appropriate conditions). The linear slope indicates that the rate of the enzymatic reaction and the increase in product formation are proportional to the enzyme reaction.
As the reaction proceeds, the substrate is consumed and the acceleration decreases. Figure 4(a) shows the time-course effect of an enzymatic reaction. The native sPLA 2 exhibited a linear rate increase over a 20 min reaction and the sPLA 2 : Q and sPLA 2 : Qn experienced a reduction in enzymatic activity of approximately 57 ± 4% and 63 ± 12%, respectively, in the same time period (Figure 4(a)). In fact, there is no statistically significant difference in Figure 4(a) between both inhibitors at 20 minutes of enzyme kinetic experiments. The data in Figure 4(a) suggest a trend towards greater Qn inhibition over Q. The results of Figure 4(a) show that the saturation of the active site of sPLA 2 in the presence of Qn already occurs after 40 minutes whereas the sPLA 2 incubated with Q, the active site of sPLA 2 is saturated after 30 minutes. These results suggest that the inhibition profile of Q to Qn is different and that these compounds have slightly different inhibition capabilities of sPLA 2 when it is purified from the Crotalus durissus terrificus venom, and the inhibitions induced by Q or Qn were statistically similar. The sPLA 2 of Crotalus durissus terrificus has been characterized as an allosteric enzyme in the presence of 4-nitro-3-(octanoyloxy)benzoic acid (NOBA or NOB), which is a chromogenic substrate specific for phospholipase A 2 [25][26][27]. Figure 4 a swelling value of 0.27 ± 0.06 mL ( = 5, and * < 0.05) and 0.32 ± 0.04 mL ( = 5, and * < 0.05) for this time interval. Within the same time interval, sPLA 2 : Q showed maximum edema of 0.18 ± 0.04 mL ( = 5, * < 0.05) and 0.28 ± 0.05 mL ( = 5, * < 0.05) in the same time interval. Furthermore, sPLA 2 : Qn showed maximum edema values of 0.18 ± 0.05 mL ( = 5, * < 0.05) and 0.023 ± 0.05 mL ( = 5, * < 0.05), respectively. These results showed that both Q and Qn significantly inhibit sPLA 2 enzyme activity, and the inhibition by Qn was two times higher than that of Q ( Figure 5(a)). Figure 5(b) shows the myotoxic activity induced by native sPLA 2 , sPLA 2 : Q, and sPLA 2 : Qn. The extent of the damage caused by sPLA 2 to skeletal muscles was assessed by quantifying the CK levels, which are widely used as an indirect marker of muscle damage. For trials with snake toxins, CK is used as a marker to assess the damage to skeletal muscles in the presence of snake venom. Three hours after the native sPLA 2 injection, the CK value was 1,230 ± 270 U/L ( = 5, * < 0.05). For the sPLA 2 : Q and sPLA 2 : Qn, the serum CK levels were 780 ± 120 U/L ( = 5, * < 0.05) and 680 ± 69 ( = 5, * < 0.05), respectively. In addition to trials with sPLA 2 s that had been chemically treated with both flavonoids, assays in which the animals were pretreated with 100 L (0.3 mM/mL, IP injection, = 5 and * < 0.05) of Q and Qn were also performed. Figure 6  terrificus. Qn exhibited a neutralizing effect that was two times higher than the effect induced by Q.

Discussion
Quercetin (Q) is considered one of the most abundant natural flavonoids and it is mainly found in fruits and other foods.
Quercetin is typically consumed in its glycosylated form as quercitrin (Qn), but multiple studies carried out with the aglycone form demonstrated its potent anti-inflammatory effect. However, the in vivo effectiveness of this compound has been questioned. The results of experiments on in vivo models of inflammation showed that Qn was more effective in reducing inflammation in comparison to Q, which showed better results in the in vitro assays [18,28,29].
Therefore, to shed some light on the inhibitory role of Qn in the inflammatory process, the effect of this flavonoid was evaluated in a typical sPLA 2 purified from the venom of C. d. terrificus by using several experimental and theoretical methods, including chromatography, circular dichroism, molecular docking, and other in vitro and in vivo biological assays. Chromatography showed that binding to Q did not change the retention time of sPLA 2 relative to sPLA 2 : Qn samples, which exhibited a longer retention time than the native sPLA 2 and sPLA 2 : Q. This finding suggests that Qn may have caused structural changes in sPLA 2 , as also indicated by the results from circular dichroism and fluorescence scanning assays. These structural changes may be caused by the molecular interactions of Qn with the C. d. terrificus sPLA 2 , which could involve hydrogen bonding, hydrophobic and electrostatic interactions between Qn and the amino acid residues Gly 30, Gly 32, His 48, and Asp 49 and the Ca 2+ ion as suggested by the molecular docking results. Indeed, crystal complexes of porcine pancreatic phospholipase A 2 /berberine (PDB ID 4DBK), Daboia russelii pulchella sPLA 2 /berberine (PDB ID 2QVD, [30]), and acidic Bothrops jararacussu sPLA 2 (BthA-I)/p-bromophenacyl bromide presented similar ligand/protein interactions to those of the C. d. terrificus sPLA 2 / Qn docking complex, that is, involving amino acids from the Ca 2+ -binding loop (e.g., Gly 30) and catalytic site (e.g., Asp 49 and His 48).
The results of the enzyme kinetic studies show that the inhibition induced by quercitrin (Qn) is not the same as that observed for quercetin (Q), and this finding is apparent after 40 minutes (Figure 4(a)). This difference in the inhibitory capacity of Q and Qn against the sPLA 2 from Crotalus durissus terrificus is supported by the results shown in Figure 4(b), which demonstrate the kinetic behavior of native sPLA 2 , of sPLA 2 with quercetin, and sPLA 2 with quercitrin. The difference between the flavonoids is most likely caused by the presence of a rhamnose sugar in Qn in accordance with the docking studies presented in Figure 3, which shows the insertion of Qn in the hydrophobic channels of sPLA 2 . Rhamnose appears to inhibit the substrate's access to the sPLA 2 catalytic site.
An analysis of the enzymatic and pharmacological tests performed with sPLA 2 and sPLA 2 that were previously treated with both flavonoids strongly suggests that the enzymatic activity of sPLA 2 is not crucial for edema or the myotoxic effects induced by sPLA 2 . The partial protein unfolding induced by Qn significantly contributes to the decrease in the edema and myonecrosis induced by native sPLA 2 but did not abolish these effects. Pretreating sPLA 2 with Qn induced more protein modifications than pretreating with Q. The changes in the pharmacological activity of the sPLA 2 that was pretreated with Q or Qn indicate that the calcium loop region may be involved in the molecular interaction between the sPLA 2 from Crotalus durissus terrificus and the receptors. In previous studies, Lambeau et al. used a Ca 2+ loop mutant derived from sPLA 2 that was isolated from venom to demonstrate the importance of this loop in the sPLA 2 interaction with the M-type receptor [31]. sPLA 2 from venom has been found to interact with a variety of mammalian sPLA 2 -binding proteins, such as Nand M-type receptors, 14-3-3 proteins and calmodulin, pentraxins and associated proteins, crocalbin, pulmonary surfactant proteins, KDR VEGF receptor 2, and factor Xa [32]. Furthermore, Rouault et al. also demonstrated that not only is the calcium binding loop region involved in binding to the receptor, but the interfacial binding domain is also involved. Thus, the stereochemical inhibition from when the substrate was binding to the active site of sPLA 2 (as induced by Qn) could explain the different degrees of inhibition for quercetin (Q) and quercitrin (Qn).
Catalytically active sPLA 2 can induce various biological and pathological effects, as in the sPLA 2 present in snake venom. Generally, PLA 2 causes these biological, physiological, and pathological activities through its enzymatic activity, which result in the increased production of arachidonic acid, which is the rate-limiting step in the generation of eicosanoids and platelet activating factors. This effect is caused by increased levels of intracellular arachidonic acid that stimulate the activity of cyclooxygenase 2 [33][34][35] and induce an increase in free radical peroxides and pro inflammatory cytokines. The increased levels of hydrogen peroxide may therefore lead to an increase in the lipid peroxidation levels, which can lead to cell membrane lesions such as those in skeletal muscle cells. Furthermore, sPLA 2 can reportedly increase the mobilization of internal calcium through an indirect mechanism. This mobilization may lead to the activation of calpain, a member of a cytoplasmic protease family that can stimulate the activity of xanthine oxidase. This activity can lead to an increase in the concentration of molecular oxygen and may further exacerbate cellular injury [36,37]. Several studies showed that Q and Qn are potent antioxidants. Results obtained by other authors demonstrated that the protective effect of these two flavonoids may be caused by their ability to neutralize the cytotoxic action of free radicals [38,39]. The difference in the levels of protective or neutralizing effects observed between Q and Qn treatments may be caused by the presence of rhamnose because the only difference between Q and Qn is the presence of this sugar.
According to Lespade et al., [40], Kim et al., [41] glycosylation may increase the antioxidant properties of flavonoids [40,41]. Moreover, Qn confers better protection than Q in some cases by protecting cells from ROS generation as well as ROS side effects [42]. The results in Figure 6 show that pretreating animals with Q and Qn can greatly reduce the toxic activity of sPLA 2 . These results also suggest that the action of these compounds occurs at the intracellular level and involves the neutralization of ROS and ROS side effects such as the activation and enhancement of the inflammation cascade. The presence of rhamnose in Qn is crucial to its protective activity against sPLA 2 from Crotalus durissus terrificus in both in vitro and in vivo studies, which indicates that quercitrin (Qn) is more effective than quercetin (Q) at the cellular level. Qn inhibits the interfacial binding domain of the sPLA 2 from Crotalus durissus terrificus from interacting with its receptor.