Research Article
An Evaluation of 3-Rhamnosylquercetin, a Glycosylated
Form of Quercetin, against the Myotoxic and Edematogenic
Effects of sPLA2 from Crotalus durissus terrificus

Daniela de Oliveira Toyama,1 Henrique Hessel Gaeta,2

Marcus Vinícius Terashima de Pinho,2,3 Marcelo José Pena Ferreira,4

Paulete Romoff,4 Fábio Filippi Matioli,5Angelo José Magro,5

Marcos Roberto de Mattos Fontes,5 and Marcos Hikari Toyama2

1 Centro de Ciências Biológicas e da Saúde (CCBS), Universidade Presbiteriana Mackenzie, 01302-907 São Paulo, SP, Brazil
2 Campus Experimental do Litoral Paulista, UNESP, Laboratório de Biologia Molecular e Pept́ıdeos, BIOMOLPEP,
11330-900 São Vicente, SP, Brazil

3 Programa de Pós-Graduação em Farmacologia, Faculdade de Ciências Médicas, UNICAMP, 13083-970 Campinas, SP, Brazil
4 Escola de Engenharia, Universidade Presbiteriana Mackenzie, 01302-907 São Paulo, SP, Brazil
5 Departamento de Fı́sica e Biof́ısica, Instituto de Biociências, UNESP, 18618-970 Botucatu, SP, Brazil

Correspondence should be addressed to Marcos Hikari Toyama; marcoshikaritoyama@gmail.com

Received 11 October 2013; Revised 9 December 2013; Accepted 9 December 2013; Published 18 February 2014

Academic Editor: Kota V. Ramana

Copyright © 2014 Daniela de Oliveira Toyama et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA
2
) 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 sPLA

2
. 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 sPLA
2
,

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 sPLA

2
, 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 sPLA

2
.

1. 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 themost 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, mam-
malian genomes encode several types of sPLA

2
-binding pro-

teins, 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 secre-

tory phospholipase A
2
s purified from humans can induce

Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 341270, 11 pages
http://dx.doi.org/10.1155/2014/341270

http://dx.doi.org/10.1155/2014/341270


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

[7–9]. This approach could be especially useful for develop-
ing 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
[10–12]. 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, conse-
quently presenting a potential method for reducing and con-
trolling 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 [15–17]. 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 pro-
nounced effect in downregulating the inflammatory response
relative to Qn, other studies highlight a significant anti-
inflammatory effect from both glycosides (Qn and ruti-
noside) in experimental colitis models in rats and other bio-
logical 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. terri-
ficus 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).

2. Material and Methods

2.1. Materials. The venom from Crotalus durissus terrificus
(C. d. terrificus) was kindly donated by the Butantan Institute
(São Paulo, Brazil). The solvents, chemicals, and reagents
used for protein purification and characterization (HPLC
grade or higher) were acquired from Sigma-Aldrich Chemi-
cals (3050 Spruce St., St. Louis, MO 63103, USA),Merck (One
Merck Drive, Whitehouse Station, NJ, USA), and Bio-Rad

(USA). Male Swiss mice (20–25 g) were obtained from the
Multidisciplinary Center for Biological Research (CEMIB) of
the State University of Campinas (UNICAMP). The animals
were maintained under standard conditions (22 ± 2∘C; 12 h
light/dark cycle), with food and water available ad libitum.
All animal experiments were performed in accordance with
Brazilian laws for the Care and Use of Laboratory Animals,
and the protocols were approved by the Committee of Ethics
from UNICAMP number 2898-1.

2.2. Purification of Quercitrin. Quercitrin (Qn) was purified
from the leaves of Baccharis microdonta DC. that were
collected in Campos do Jordão, SP, in June 2008. A voucher
specimen has been deposited at the Herbarium of Prefeitura
Municipal de São Paulo (PMSP) under number 8980. Dried
and powdered leaves (241 g) were defatted with n-hexane
and subsequently extracted with methanol at room tem-
perature. Following its concentration under a vacuum, the
crude MeOH extract (97.5 g) was suspended in MeOH :H

2
O

(1 : 1), 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 (108.8mg) 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].

2.3. Purification of Phospholipase A2. To purify theC. d. terri-
ficus sPLA

2
, whole venomwas first fractionated as previously

described by [20]. Dried venom (45mg) was dissolved in
ammonium bicarbonate buffer (0.2M, pH 8.0) and clarified
by centrifugation (4,500×g, 1min). 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 0.2mL/min for the elution
of fractions. The absorbance was monitored at 280 nm. The
separated crotoxin-like fractionwas immediately lyophilized.
The lyophilized fraction was then subjected to reverse-phase
chromatography using a 𝜇-Bondapak C18 column (0.39 ×
30 cm) with a flow rate of 1mL/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].

2.4. Treating sPLA
2
with Quercetin and Quercitrin. The incu-

bations 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 𝜇L of
0.1mM solution) was added to 400 𝜇L of a homogenized,



BioMed Research International 3

purified C. d. terrificus sPLA
2
solution (1mg/mL). The mix-

ture was then incubated for 90min at room temperature,
and 200𝜇L aliquots were loaded into a preparative reverse-
phase 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 1.0mL/
min. The chromatographic run was monitored at 214 nm.

2.5. Circular Dichroism Spectroscopy. The secondary struc-
ture 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 theCD signals
they produce are sensitive to the overall tertiary structure of
the protein.

In this study, we used both assay types to evaluate the sec-
ondary 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 10mM

sodium phosphate buffer (pH 7.4), and the final protein con-
centrations were adjusted to 8.7mM. This protein solution
was then subjected to centrifugation at 4,000×g for 5min,
and the resulting supernatant was transferred to a 1mmpath-
length quartz cuvette. Circular dichroism spectra within a
wavelength range of 185–300 nmwere acquired in-housewith
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 sur-
rounding 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.

2.6. Molecular Docking. For quercitrin (Qn) in silico design
and docking simulations, the Avogadro v.0.9.4 (http://avo-
gadro.openmolecules.net/) programwas 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 10 Å
radius around the His48 residue located at the catalytic site

of monomers A and C of the C. d. terrificus sPLA
2
crystallo-

graphic structure. Additionally, other cavities on the protein
surface were tested to identify other potential docking sites.
The N𝛿1 atoms from the catalytic histidine of the C. d.
terrificus sPLA

2
crystallographicmodel were protonated, and

the simulations generated approximately 1000 docking solu-
tions 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 protein-
ligand 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].

2.7. 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 20min. All assays were performed by
using 𝑛 = 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
(20𝜇g),

the reaction mixture was incubated for 40min at 37∘C, and
the absorbance was read at 10min intervals. The effect of
the substrate concentration on enzyme activity was deter-
mined by measuring the absorbance increase after a 20min
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 andQn
were dissolved in 1% DMSO.

2.8. 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 𝜇g 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).



4 BioMed Research International

2.9. 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 bothQ andQn.These samples were injected into
the left gastrocnemius muscle of male Swiss mice (18–20 g;
𝑛 = 5). The right gastrocnemius muscle was injected with
50 𝜇L of 0.5mg/mL sPLA

2
samples. Control mice received

an equal volume of 0.15M 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 𝜇L of plasma, which was incubated for 3 minutes at 37∘C
with 1.0mL of the reagent according to the kit protocol. The
resulting activity was expressed in U/L.

2.10. Statistical Analysis. Results are reported as the means ±
SEM of replicated experiments. The significance of differ-
ences 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%.

3. Results

3.1. 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, whichwas the same as that found

by [21]; this finding shows that the methods used here were
stable, and they generated reliable and accurate data. Further-
more, the analysis result of sPLA

2
mass spectrometry: Qnwas

14580.90, so thismass is the product of sPLA
2
incubationwith

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).

3.2. Circular Dichroism Analysis. Figure 2 shows the circu-
lar 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 nmwas 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

2.0

1.6

1.2

0.8

0.4

0.0

0 10 20 30 40 50 60

Time (min)

100

80

60

40

20

00

A
2
1
4

nm

Bu
ffe

r B
 (%

)

Buffer B (%)

sPLA2

A214 nm

sPLA2 : Qn

sPLA2 : Q

(a)

100

90

80

70

60

50

40

30

20

10

00
12000 13000 14000 15000 16000 17000

m/z

In
te

ns
ity

 (%
)

14132.5 14580.9
sPLA2 sPLA2 : Qn

(b)

Figure 1: Purification and chemical modification of secretory
phospholipase A

2
(sPLA

2
). A fractionation of the whole venom was

performed by reverse-phase HPLC (C5 column, 0.10 cm × 25 cm)
using a nonlinear concentration gradient of buffer to obtain a high-
purity protein. (a) shows a comparative profile of native sPLA

2
,

sPLA
2
: Q, and sPLA

2
: Qn when subjected to reverse-phase HPLC.

(b) shows the MALDI-TOF mass spectrometry analysis of native
sPLA

2
and sPLA

2
: Qn, indicating the difference in the molecular

mass corresponding to one molecule of bound quercitrin.

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
.

3.3. Molecular Docking of sPLA
2
with Compounds. In addi-

tion to chromatographic and biophysical experiments, dock-
ing studies were also performedwith theCro crystallographic



BioMed Research International 5

25

20

15

10

05

00

−05

−10

−15

CD

190 200 210 220 230 240 250 260

Wavelength (nm)

sPLA2

sPLA2 : Qn

sPLA2 : Q

(a)

CD

Wavelength (nm)

2.0

1.0

0.0

−1.0

−2.0

−3.0

260 270 280 290 300 310 320

sPLA2

sPLA2 : Qn

sPLA2 : Q

(b)

Figure 2: The far-UV (ultraviolet) CD spectrum of proteins can
reveal important characteristics of their secondary structure. (a)
shows the results of CD spectra from native sPLA

2
, sPLA

2
: Q, and

sPLA
2
: Qn. Data from 185–280 nm are shown. The CD spectra are

expressed in theta machine units in millidegrees. The near-UV
CD spectrum (>250 nm) of proteins provides information on the
tertiary structure. The signals obtained in the 250–300 nm region
are 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 amino acids. (b)
shows the near-UVCDspectrumof the native sPLA

2
, sPLA

2
: Q, and

sPLA
2
: Qn.

model [22], and they were used to analyze the probable pref-
erential 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 theC. 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 10 Å radius around the
His48 residue, which was located at the catalytic site of the A
andCmonomers of theC. d. terrificus sPLA

2
crystallographic

structure. Additionally, other cavities on the protein surface
were also tested to identify other potential docking sites.
The N𝛿1 atoms from the catalytic histidine in the C. d.
terrificus sPLA

2
crystallographicmodel were protonated, and

the simulations generated approximately 1000 docking solu-
tions to provide a representative population. The remaining
docking parameters were defined according to the GOLD
v.5.0.1 default settings.Thedocking solutions betweenQn 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 Ca2+ ion.

3.4. 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 20min 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 greaterQn 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 30minutes.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
[25–27]. Figure 4(b) shows the

substrate effects on the sPLA
2
activity, and the native sPLA

2

exhibited a Vmax value of 0.254 ± 0.09 and a Km value of
0.08 ± 0.002, whereas sPLA

2
: Qn and sPLA

2
: Q had Vmax

values of 0.12 ± 0.03 and 0.10 ± 0.03, and Km values of
0.04 ± 0.002 and 0.051 ± 0.004, respectively.

3.5. Pharmacological Assays. The native sPLA
2
had a max-

imum edema value of approximately 30 to 60min, with



6 BioMed Research International

(a)

ALA-23

GLY-32

GLY-30

Ca

ASP-49

HIS-48

(b)

(c) (d)

Figure 3: Structural representation ofQbound to sPLA
2
fromdocking simulations. (a) shows a cartoon representation of the sPLA

2
structure.

quercetin is shown in a stick representation, and the Ca2+ ion is represented as a blue sphere. (b) shows the quercetin molecule and its main
amino acid interactions. (c) shows a surface representation of sPLA

2
bound to a quercetin molecule (stick representation). (d) shows the dot

representation of the Quercetin molecule in the bound position. Two amino acid residues (Asp49 and His48) from sPLA
2
are represented as

sticks.

a swelling value of 0.27 ± 0.06mL (𝑛 = 5, and ∗𝑃 < 0.05)
and 0.32 ± 0.04mL (𝑛 = 5, and ∗𝑃 < 0.05) for this time
interval. Within the same time interval, sPLA

2
: Q showed

maximum edema of 0.18 ± 0.04mL (𝑛 = 5, ∗𝑃 < 0.05) and
0.28 ± 0.05mL (𝑛 = 5, ∗𝑃 < 0.05) in the same time interval.
Furthermore, sPLA

2
: Qn showed maximum edema values of

0.18 ± 0.05mL (𝑛 = 5, ∗𝑃 < 0.05) and 0.023 ± 0.05mL
(𝑛 = 5, ∗𝑃 < 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 amarker 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 ± 270U/L

(𝑛 = 5, ∗𝑃 < 0.05). For the sPLA
2
: Q and sPLA

2
: Qn, the

serum CK levels were 780 ± 120U/L (𝑛 = 5, ∗𝑃 < 0.05) and
680 ± 69 (𝑛 = 5, ∗𝑃 < 0.05), respectively.

In addition to trials with sPLA
2
s that had been chemically

treatedwith both flavonoids, assays inwhich the animalswere
pretreated with 100 𝜇L (0.3mM/mL, IP injection, 𝑛 = 5 and
∗
𝑃 < 0.05) of Q and Qn were also performed. Figure 6(a)
shows the edema of animals pretreated with both flavonoids.



BioMed Research International 7

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Vo
 (4

2
5

nm
)

0 10 20 30 40

Time (min)
sPLA2

sPLA2 : Qn
sPLA2 : Q

(a)

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Vo
 (A

4
2
5

nm
)

0.00 0.03 0.06 0.09 0.12 0.15 0.18 0.21

Substrate (mmol/mL)

sPLA2

sPLA2 : Qn
sPLA2 : Q

(b)

Figure 4: (a) shows the results of the enzymatic activity assays
that were performed by using a synthetic chromogenic substrate for
PLA
2
(NOBA). The reaction was monitored at 425 nm. sPLA

2
: Q

and sPLA
2
: Qn exhibited a significant decrease in activity when

compared to native sPLA
2
. (b) shows the effect of the substrate

concentration on enzyme activity in the presence of native sPLA
2
,

sPLA
2
: Q, and sPLA

2
: Qn.

The results of the edema assay for animals receiving 0.9%
saline (100𝜇L, IP injection, 𝑛 = 5 and ∗𝑃 < 0.05) were 0.38 ±
0.06mL (𝑛 = 5 and ∗𝑃 < 0.05) and 0.44 ± 0.05mL (𝑛 = 5 and
∗
𝑃 < 0.05) at 30min and 60min into the edema time-course
experiment after the injection of native sPLA

2
, respectively.

The animals that received Q (30󸀠) had 0.28 ± 0.03mL edema
values at 30min (𝑛 = 5, ∗𝑃 < 0.05) and 0.38 ± 0.08mL at
60min (𝑛 = 5, ∗𝑃 < 0.05). The animals treated with Qn

0.4

0.3

0.2

0.1

0.0

0 30 60 90 120 150 180 210 240

Time (min)

Ed
em

a (
m

L)

∗

∗
∗

∗

sPLA2

sPLA2 : Qn
sPLA2 : Q

(a)

∗

∗

1000

800

600

400

200

000

CK
 (U

/L
)

sPLA2 sPLA2: Q sPLA2: Qn

(b)

Figure 5: (a) shows the results of paw edema that was induced
after the injection of sPLA

2
, sPLA

2
: Q, and sPLA

2
: Qn into the right

paws of Swiss mice. Measurements were made after 30, 60, 120, and
240min, and all the edema results expressed in (a) were obtained by
subtracting the saline injection values. (b) shows the myonecrosis
levels as evaluated by CK levels in Swiss mice. Fifty micrograms of
native sPLA

2
, sPLA

2
: Q, and sPLA

2
: Qn at a final concentration of

0.5mg/mL were injected into the gastrocnemius muscle.The results
are expressed as units of enzymatic activity per liter (U/L). Error bars
indicate the SEM. ∗𝑃 < 0.05 compared to native sPLA

2
.

(30󸀠) had swelling times of 0.21 ± 0.04mL at 30min (𝑛 = 5,
∗
𝑃 < 0.05) and 0.23 ± 0.09mL at 60min (𝑛 = 5, ∗𝑃 < 0.05).

Figure 6(b) shows the effects of injecting Q and Qn into
animals 30min before sPLA

2
administration, which were

injected into the left gastrocnemius muscle of male Swiss
mice.The group that received saline (control group) exhibited
CK levels of 970 ± 156U/L (100 𝜇L, IP injection, 𝑛 = 5, ∗𝑃 <
0.05). The group that received Q and Qn showed a plasma
CK level of 870 ± 96U/L (100 𝜇L, IP injection, 𝑛 = 5, ∗𝑃 <
0.05) and 380 ± 122U/L (100 𝜇L, IP injection, ∗𝑃 < 0.05),
respectively. The Qn injected into the animals 30min before
sPLA
2
was able to significantly reduce themyotoxic effect that

was induced by the sPLA
2
isolated from Crotalus durissus



8 BioMed Research International

∗

∗
∗

∗

∗

∗

Q (30󳰀)
Qn (30󳰀)

0.5

0.4

0.3

0.2

0.1

0.0

0 30 60 90 120 150 180 210 240

Time (min)

Ed
em

a (
m

L)

sPLA2

(a)

∗

∗

CK
 (U

/L
)

1400

1200

1000

800

600

400

200

000

Q (30󳰀) Qn (30󳰀)sPLA2

(b)

Figure 6: (a) shows the results from paw edema in the animals that
were injected with quercitrin (Qn 30󸀠) and quercetin (Q 30󸀠) 30min
before sPLA

2
administration into the right paw of Swiss mice. The

control group received a saline injection prior to the administration
of sPLA

2
. Measurements were made after 30, 60, 120, and 240min,

and all edema results expressed in (a)were obtained after subtracting
the edema values from the saline injection. (b) shows the results of
paw edema in animals that were injected with quercitrin (Qn 30󸀠)
or quercetin (Q 30󸀠) 30min before the administration of sPLA

2
.

The control group received saline. Myonecrosis was evaluated on
the basis of CK levels after 50mg of native sPLA

2
was injected at

a final concentration of 0.5mg/mL into the gastrocnemius muscle.
The results are expressed as units of enzymatic activity per liter
(U/L). Error bars indicate the SEM. ∗𝑃 < 0.05 compared to native
sPLA

2
.

terrificus. Qn exhibited a neutralizing effect that was two
times higher than the effect induced by Q.

4. Discussion

Quercetin (Q) is considered one of themost 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 theoret-
ical 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 indi-

cated 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 betweenQn and the amino acid
residues Gly 30, Gly 32, His 48, and Asp 49 and the Ca2+ ion
as suggested by themolecular 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 lig-
and/protein interactions to those of theC. d. terrificus sPLA

2
/

Qn docking complex, that is, involving amino acids from the
Ca2+-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 enzy-
matic 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

withQn inducedmore proteinmodifications thanpretreating
with Q. The changes in the pharmacological activity of



BioMed Research International 9

the sPLA
2
that was pretreated with Q or Qn indicate that the

calcium loop region may be involved in the molecular inter-
action between the sPLA

2
from Crotalus durissus terrificus

and the receptors. In previous studies, Lambeau et al. used a
Ca2+ 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 N-

and M-type receptors, 14-3-3 proteins and calmodulin, pen-
traxins and associated proteins, crocalbin, pulmonary sur-
factant 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, physiologi-

cal, 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 [33–35] and
induce an increase in free radical peroxides and pro inflam-
matory 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 themobilization of internal calcium through an indi-
rect mechanism.This mobilization may lead to the activation
of calpain, amember 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 thatQ andQnare 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 betweenQ andQn treatmentsmay 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] glycosy-
lation 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
fromCrotalus durissus terri-

ficus 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.

Conflict of Interests

The authors have no conflict of interests to disclose.

Acknowledgments

The authors are grateful to the Coordenadoria de Aperfeiçoa-
mento de Pessoal de Nı́vel Superior (CAPES), the Fundo
Mackenzie de Pesquisa, the Conselho Nacional de Desen-
volvimento Cient́ıfico e Tecnológico (CNPq), and the Fun-
dação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) for their financial support (FAPESP Proc. nos.
2011/06704-4, 2012/06502-5, and 2013/12077-8) and to the
Instituto Nacional para Pesquisa em Toxinas (INCT-Tox).

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