Design and Synthesis of Butenolide-based Novel Benzyl Pyrrolones: Their TNF-α based Molecular Docking with in vivo and in vitro Anti-inflammatory Activity

Article abstract of DOI:10.1111/cbdd.12522

A focused library of novel benzyl pyrrolones has been synthesized and their in silico molecular docking studies carried out against TNF-α target. Among all the docked molecules, compound 3f showed best glide score of -6.89. All the synthesized compounds were evaluated for in vivo anti-inflammatory activity by carrageenan-induced paw edema model. Compounds showing significant anti-inflammatory activity were further tested for their in vitro TNF α expression. Compounds 3b and 2b were found to show significant inhibition of 76.22% and 71.47%, respectively after 5 h in comparison with standard drug indomethacin, which showed 80.98% inhibition of inflammation. Compounds 3b and 2b also suppressed TNF α level by 65.03% and 60.90% as compared indomethacin, which showed 68.84% of inhibition. Compound 3b showed significant analgesic activity of 60.04%, and its activity was comparable with indomethacin (64.04%). Compounds 3b and 2b were also tested for their effect on protein expression of COX-2 and NF-κB in the liver tissues. Compounds 3b and 2b were further evaluated for their gastric risk and lipid peroxidation action and showed superior GI safety along with reduction of LPO as compared to indomethacin. Hepatotoxicity study showed that these two compounds did not cause any damage to liver.

Full text of DOI:10.1111/cbdd.12522

Received Date : 23-May-2014
Revised Date : 17-Sep-2014
Accepted Date : 05-Jan-2015
Article type : Research Article
Design and synthesis of Butenolide Based Novel Benzyl Pyrrolones: Their
TNF-α Based Molecular Docking with in-vivo and in-vitro Anti-inflammatory
Activity
a
a
a
b
a
Yakub Ali , Mohammad Sarwar Alam* , Hinna Hamid , Asif Husain , Syed Shafi , Abhijeet
c
d
a
a
a
a
Dhulap , Firasat Hussain , Sameena Bano , Chetna Kharbanda , Syed Nazreen , Saqlain Haider
a
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New
Delhi-110 062, India
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), New Delhi-110 062, India
c
CSIR Unit for Research and Development of Information Products, Pune -411038, India
d
Department of Chemistry, Faculty of Science, Delhi University, New Delhi, India
*
Correspondence to Prof. Mohammad Sarwar Alam, Email ID: msalam@jamiahamdard.ac.in;
msalam5555@gmail.com Phone: +91 11 26059688 (5555), Fax:+91 11 26059663
A focused library of novel benzyl pyrrolones has been synthesized and their in silico molecular
docking studies carried out against TNF-α target. Among all the docked molecules, compound 3f
showed best glide score of -6.89. All the synthesized compounds were evaluated for in vivo anti-
inflammatory activity by carrageenan induced paw edema model. Compounds showing
significant anti-inflammatory activity were further tested for their in vitro TNF α expression.
Compounds 3b and 2b were found to show significant inhibition of 76.22% and 71.47%
respectively after 5 h in comparison to standard drug indomethacin which showed 80.98%
inhibition of inflammation. Compounds 3b and 2b also suppressed TNF α level by 65.03% and
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through the copyediting, typesetting, pagination and proofreading process which may lead to
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6
0.90% as compared indomethacin which showed 68.84% of inhibition. Compound 3b showed
significant analgesic activity 60.04% and its activity was comparable with indomethacin
64.04%). Compounds 3b and 2b were also tested for their effect on protein expression of COX-
and NF-κB in the liver tissues. Compounds 3b and 2b were further evaluated for their gastric
(
2
risk and lipid peroxidation action and showed superior GI safety along with reduction of LPO as
compared to indomethacin. Hepatotoxicity study showed that these two compounds did not
cause any damage to liver.
Keywords: benzyl pyrrolones, anti-inflammatory, TNF-α, anti-nociceptive, ulcerogenic,
hepatotoxicity, immunohistochemistry.
Butenolides, a family of α, β-unsaturated lactones, also known as furanones, are ubiquitous
chemical moieties. They are found in many natural products such as Digitoxigenin
Goniobutenolides A & B and lignans (1). Physiological activity of the natural lactones is well
known since ancient time. For example, santonin was used as an important anthelmintic and
ascaricidal agent (2, 3). Butenolides have been reported to possess important biological activities
such as antibacterial, anticonvulsant, anti-inflammatory, analgesic, antitumor, antiviral and
anticancer activity (4, 5-9). γ-Lactone ring present in the butenolide derivatives is significantly
reactive and it could be utilized for the synthesis of nitrogen heterocycle analogs such as benzyl
pyrrolone derivatives which shows diverse biological activities (10, 11). A series of new
butenolide derivatives having potential anti-inflammatory and analgesic activities with reduced
gastric ulceration has been reported earlier (12-14). In order to further explore the chemical
diversity, we have synthesized a new series of twenty four benzyl pyrrolone derivatives based on
butenolide moiety with potent anti-inflammatory activity with reduced side effects. All the
synthesized compounds were evaluated for their anti-inflammatory activity. In order to explore
the mechanistic aspects of activity, the synthesized molecules were subjected to in silico
molecular docking studies with respect to TNF α target. The compounds showing significant
anti-inflammatory activity were further evaluated for their effect on in-vitro TNF-α level. These
compounds were then tested for gastric ulceration, LPO and hepatotoxcity. The active
compounds were also assessed for their effect on protein expression of NF-κB and COX-2 in
carrageenan induced hepatic tissue by immnohistochemistry.
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Experimental protocols
Biological evaluations
Drugs and Chemicals
Indomethacin, carrageenan, carboxymethylcellulose, trichloroacetic acid and thiobarbituric acid
were purchased from Sigma-Aldrich Chemicals Pvt. Limited, Bangalore, India and ELISA kits
of TNF-α was purchased from eBioscience (San Diego, California).
Anti-inflammatory activity
The anti-inflammatory activity was carried out by the reported method (15). Animals were
divided into twenty six groups of five animals each. Acute paw edema was induced by sub-
plantar injection of 0.1% freshly prepared carrageenan suspension in normal saline into the right
hind paw of each rat after 1 hour of administrating the test samples. The negative control was
administered with 0.5% carboxymethyl cellulose solution. The animals of positive control were
orally administered with standard indomethacin (20mg/kg/po). The test groups were
administered orally at dose of 20mg/kg body weight. Right hind paw volume was measured at 3
h and 5 h after carrageenan injection with the help of digital plethysmometer. The percent anti-
inflammatory activity was calculated according to the formula given below.
%
Anti-inflammatory Activity = [V -V /V ] x 100
C t C
where, V represents the mean increase in paw volume in rats treated with test sample and V
t
C
represents the mean increase in paw volume in control group of rats.
Molecular Docking studies on TNF-α
Crystallized structure of 2AZ5 was chosen from Protein Data Bank and used as target for
molecular docking studies. 2AZ5 structure was imported in Schrodinger using Protein
preparation wizard. Missing hydrogen and atoms were added using prime interface. Undesired
water molecules were removed. The protein was then optimized and minimized to give low
energy and structurally correct target protein. As the target protein had already the site for
reference ligand, the grid was generated by selecting the ligand as the reference ligand. Finally
the grid was validated and was used for further docking with new unknown ligands to predict
their docking score. Chemical structures were drawn in maestro and geometrically refined by
LigPrep module. In this module 2-D structures were converted into 3-D structures, which were
further subjected to OPLS-2005 force field to generate single low energy 3-D structure for each
input structure. During this step chiralities were maintained. Docking was carried out using Glide
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software. It was carried out using extra precision and write XP descriptor information. This
generates favorable ligand conformations which are subjected to evaluation and minimization of
grid approximation. Scoring is then carried on energy minimized conformations to generate
Glide score. Glidescore is scoring function based on empirically based Chem Score function of
Eldridge et al. It is more modified and expanded version of the Chem Score scoring function and
is used to predict binding affinity and rank the ligands. It takes account of ligand atom- receptor
atom interaction, Hydrogen bond interaction, Vander-walls interactions and solvation effects.
Glide energy refers to Modified Coulomb-van der Waals interaction energy possessed by the
ligand in the docked pose (16).
In-vitro TNF-α assay
RAW 264.7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin
(
100 units/ml), and streptomycin sulfate (100m g/ml) in a humidified atmosphere of 5% CO2.
The cells were harvested with trypsin-EDTA and diluted to a suspension in fresh medium. The
5
cells were then seeded in 96-well plates with 2.1 × 10 cells/well, and allowed to adhere for 1 h.
The medium was then induced with 100 μg/ml LPS (lipopolysaccharide), test samples (20µM),
and incubated for 24 h. The supernatant (50 μL) was then transferred into a 96-well ELISA plate
and TNF-α level were quantified by ELISA kits according to the manufacturer’s instructions.
Analgesic Activity
The writhing test in swiss albino mice was carried out using the previously reported method (17).
Swiss albino mice (35–40 g) of either sex were divided into different groups of five animals
each. Group I was taken as control and received CMC suspension only, group II received
standard drug indomethacin and rest of the groups were orally administered with synthesized
compounds at a dose of 20mg/kg. After 30 min of test samples administration, 0.6% acetic acid
solution was given to mice intraperitoneally. The number of muscular contractions was counted
over a period of 10 min after acetic acid injection. The data represents the total number of
writhes observed during 10 min and is expressed as writhing numbers.
Ulcerogenic activity
The compounds showing good anti-inflammatory activity were further evaluated for their
ulcerogenic activity. The ulcerogenic study was carried out by reported method (18). Oral
administration of test samples and standard drug (indomethacin) were given at a dose of 60
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mg/kg b.w. which is thrice the dose used for anti-inflammatory activity. Control rats received
vehicle (suspension of 1% methyl cellulose) only. Animals were sacrificed 5h after dose
administration.
Lipid peroxidation activity
Previously reported method (19) was followed for evaluating LPO activity. The gastric mucosa
was scraped with two glass slides, weighed (100 mg) and homogenized in 1.8 ml of 1.15% ice
cold KCl solution. One ml of suspension medium was taken from the supernatant, followed by
the addition of 0.5 ml of 30% trichloroacetic acid and 0.5 ml of 0.8% thiobarbituric acid reagent.
The tubes were covered with aluminum foil and kept in a shaking water bath for 30 min at 80°C.
After 30 min, tubes were taken out and kept in ice cold water for 10 min. These were then
centrifuged at 3000rpm for 15 min. The absorbance of supernatant was read at 540 nm at room
temperature against the blank.
Hepatotoxicity study
For this study, the dosing pattern was kept same as that for ulcerogenic studies. The experimental
rats were sacrificed after 5h of the administration of the test samples and standard drug and their
liver specimens were removed and stored in 10 % formalin solution (20).
Immunohistochemistry
The liver tissues were fixed in formalin and embedded in paraffin. Sections of 5 µm thickness
were cut onto poly-lysine coated glass slides. Sections were de-parafinized three times (5 min) in
xylene followed by dehydration in graded ethanol and finally rehydrated in running tap water.
For antigen retrieval, sections were boiled in 10mM citrate buffer (pH 6.0) for 5-7 min. Sections
were incubated with hydrogen peroxide for 15 min to minimize non-specific staining and then
rinsed three times (5 min each) with 1X PBST (0.05% Tween-20). Blocking solution was applied
for 10 min then sections were incubated with diluted (1:100) primary antibodies, purified rabbit
polyclonal anti-NF-κB antibody (BioLegend) and rabbit polyclonal anti-COX-2 antibody (Bio
o
Vision), overnight at 4 C in humid chamber. Further processing was done according to the
instructions of Ultra Vision plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use)
staining kit (Thermo scientific system). The peroxidase complex was visualized with 3, 3’-
diaminobenzidine (DAB). Lastly the slides were counterstained with haematoxylin, cleaned in
xylene, dehydrated with ethanol and then DPX mounting microscopic (BX 51 Olympus) analysis
was done at 40 X magnifications (21)
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Result and Discussions
Chemistry
2
-Arylidene-4-(4-fluoro-phenyl)-but-3-en-4-olides were synthesized by reacting 3-(4-fluoro-
benzoyl)-propionic acid with aromatic aldehydes in presence of sodium acetate in acetic
anhydride. Different 3-arylidene-5-(4-fluoro-phenyl)-2(3H)-1-benzyl-2(3H)-pyrrolones (1a-1h,
2
a-2h, 3a-3h) were prepared by reacting butenolides with different benzylamines in dry benzene
to give γ-ketobenzylamides, which were then lactamized in HCl to give the corresponding N-
benzyl pyrrolones. The structural confirmation of target compounds (1a-1h, 2a-2h, 3a-3h) was
1
13
done by H NMR, C NMR and Mass spectral data as well as elemental analysis. (Scheme-1)
1
The H NMR spectra showed two singlets for one proton each at δ 6.5 to 6.8 and δ 7.4 to 7.9 for
pyrrolone ring hydrogen and the olefinic hydrogen of the arylidene substituent respectively. A
singlet for two protons at δ 4.89 to 4.94 was assigned to the benzyl ring protons. Other peaks
1
3
were observed at appropriate δ values. In the C NMR spectral data, benzyl carbon appeared at δ
44.50 to 44.90. Other aromatic carbon peaks were observed at appropriate places. ESI-MS of all
+
the compounds showed [M] , [M+1] with reasonable intensity. The most active compound (3b)
was also subjected to X-ray crystallography study which is being reported for the first time here.
Anti-inflammatory activity
All the synthesized compounds were evaluated for in-vivo anti-inflammatory activity by
carrageenan-induced hind paw edema method. Among the syntheized compounds, compound
3
b exhibited maximum anti-inflammatory activity of 70.29% at 3h and 76.22% at 5h
respectively which was comparable to that of standard drug indomethacin which showed
inhibition of 74.33% at 3h and 80.98% at 5h at a dose of 20mg/kg respectively. Compound 1b
(
71.39%), 2b (71.47%), 2c (66.71), 2f (66.24), 3c (71.31%), 3f (68%), and 3g (65.53%)
showed good activity at 5h. Results are presented in table 1.
In silico docking studies
The synthesized compounds were docked for in silico study against TNF alpha target. Molecular
docking studies provided insights of molecular binding modes of molecules inside the large
pocket of TNF alpha receptor. Crystallized structure of 2AZ5 was chosen from protein data bank
and used as a target for molecular docking studies with the specific ligand indomethacin which
inhibits it. In order to analyze the binding pattern and energies of new molecules, they were
docked individually against the generated grid. The synthesized compounds docked against the
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grid showed good binding energies ranging from -32.87 to -42.97 kcal/ mol. Among all the
synthesized molecules, the most promising molecules were 3f, 3g, 2g, 2f, and 2e with the glide
score -6.89, -6.71, -6.68, -6.66, and -6.57 respectively whereas the glide score of other reference
ligand (standard indomethacin) was -5.02. The compound 3f was found to show π- π stacking
with TYR 59, TYR 151 and TYR 119 residues (fig.1a). Indomethacein was aligned perfectly
with the hydrophobic pocket of the TNF-α protein and showed π- π stacking with TYR 59 only
(fig.1b). 3f molecule (yellow colour) and Indomethacin (Red colour) are bound in the same
protein cavity are shown in fig.1c and 3D representation of all the synthesized molecules binding
in the same cavity along with indomethacin (yellow) as the reference molecule are shown in fig
.
1d. The glide score and binding energies of all the synthesized compounds are shown in table 2.
In-vitro TNF-α assay
Out of 24 compounds, eight compounds showing significant anti-inflammatory activity were
tested for their effect on in vitro TNF-α level in LPS induced RAW 264.7 cell lines. LPS acts as
a prototypical endotoxin which promotes the pro-inflammatory cytokine (IL-1, IL-6 or TNF-α)
in many cells and LPS significantly increases the level of TNF-α in RAW 264.7 cell lines. The
results of active compounds 1b, 2b, 2c, 2f, 3b, 3c, 3f and 3g are shown in fig.2. It was observed
that the active compounds decreased the TNF-α concentration as compared to control group.
Compound 3b and 2b decreased TNF- α concentration significantly by 65.03%, and 60.90% in
comparison to standard drug indomethacin which showed 68.84% inhibition. Compounds1b, 2c,
2
f, 3c, 3f and 3g showed moderate inhibition of TNF-α concentration. From the results, it is clear
that compounds 3b and 2b showing significant in vivo anti-inflammatory activity also
significantly decreased the level of TNF-α in LPS induced RAW 264.7 cell line.
Analgesic activity
The compounds that showed significant anti-inflammatory activity were also evaluated for their
analgesic activity by acetic acid induced writhing test. Acetic acid induced writhing test is used
for detecting both central and peripheral analgesia. Intraperitoneal administration of acetic acid
releases prostaglandins and sympathomimetic system mediators like PGE2 and PGF2α and their
levels are increased in the peritoneal fluid of the acetic acid induced mice (22). Among these
compounds, compound 3b showed maximum analgesic activity (60.04) and its activity was
comparable with the standard drug indomethacin (64.04%) at a dose of 20 mg/kg b.w.
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Compound 1b, 2b, 3c and 3f also showed significant analgesic activity (47.48%-55.64%). The
results are shown in fig 3.
Ulcerogenic activity
The compounds, which showed significant anti-inflammatory activity, were screened for their
ulcerogenic activity. Compounds 2b, 3b, 3c, and 2f did not cause any gastric ulceration and
disruption of epithelium cells at the given oral dose. Whereas compounds 2c and 1b caused some
slight damage in surface epithelium which was less than that of standard drug indomethacin.
Stomach wall of indomethacin treated group at low power (10X) photomicrograph showed
damage of the mucosa and sub mucosa. Stomach wall of the same section at high power (40X)
photomicrograph showed some loss of epithelial cells from the superficial and deep layers of the
mucosa. (fig.4)
Lipid peroxidation assay
The compounds that showed significant anti-inflammatory activity were further evaluated for
lipid peroxidation activity measured in terms of nmol of MDA per 100 mg of gastric mucosa
tissue. The results are shown in fig. 5. An important marker for the cellular membrane disruption
is lipid peroxidation and it is reported to proceed by free radical chain reactions. However this
process can be presented by enzymatic and non enzymatic antioxidants. Lipid peroxidation is
found to be increased in the carrageenan induced animals, however, the level of lipid per
oxidation was found to be greatly reduced in presence of the standard drug indomethacin and
active compounds 1b, 2b, 2c, 2f, 3b and 3c. Among all these compounds, compounds 3b, 3c, 2b,
and 2f caused significant reduction in lipid peroxidation as compared to standard drug whereas,
the compounds 1b and 2c caused slightly less reduction as compared to standard drug.
Hepatotoxicity studies
The results of liver histopathology studies of compounds 1b, 2b, 3b, 3c, 2c and 3c are shown in
fig. 6. It was observed that the compounds 2b, 3b, 3c, and 2f did not cause any damage to the
liver as compared to standard drug indomethacin which caused significant inflammation to the
portal, centrizonal vein and sinusoidal dilation. Furthermore the compound 1b and 2c caused
slight inflammation to the portal vein and sinusoidal vein which was less than standard drug.
Immunohistochemistry
Several mediators like COX-2, NF-κB, iNOS are activated during the process of inflammation.
Therefore, the effect of active compound 3b and 2b on hepatic expressions of COX-2 and NF-κB
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in liver tissue was also observed. The results were shown in the figures 7a & 7b respectively. It
was found that COX-2 and NF-κB expression in the animal liver tissue are increased by
carrageenan. Carrageenan treated animals in groups II showed more intense brown color which
clearly indicates increased number of cells with COX-2 and NF-κB expression as compared to
that of normal control group (I). The treatment of animals with indomethacin and active
compounds 3b and 2b reduced the number of cells showing expression of these proteins.
However this compound reduced expression better as compared to standard drug.
Crystrallographic study
Intensity data were collected at 183(2) K an Oxford XcaliburSapphire 3 diffractometer (a single
wavelength Enhance X-ray source with MoK radiation, λ = 0.71073 Å) (23). The selected
α
suitable single crystals were mounted using paratone oil on the top of a glass fiber fixed on a
goniometer head and immediately transferred to the diffractometer. Pre-experiment, data
collection, data reduction and analytical absorption corrections (24) were performed with the
Oxford program suite CrysAlisPro (25). The crystal structures were solved with SHELXS-97(26)
using direct methods. The structure refinements were performed by full-matrix least-squares on
2
F with SHELXL-97 (27). All programs used during the crystal structure determination process
are included in the WINGX software (24).
The chemical formula and ring labeling system is shown in fig.8.(compound 3b) Crystal data for
compound 3b: C24 H18 F N O , Mr, 342.80; system, triclinic; space group, P-1; unit cell
3
1
1
dimensions, a = 5.4220(3) Å; b = 12.9392(14)Å; c = 14.7576(17) Å; α = 114.702(11)⁰; β
3
=
94.904(10)⁰; γ = 91.987(9)⁰; V = 943.32(19)Å ; Z = 2; T = 298 K; R , 0.0687; R(all),
int
3
3
0
.1218;Gof = 1.039; Δρmax = 0.37 e Å ; Δ = -0.42 e Å .
ρmin
All hydrogen atoms were calculated after each cycle of refinement using a riding model, with C-
H = 0.93 Å + U (H) = 1.2U (C) for aromatic H atoms, with C-H = 0.97 Å + U (H) =
iso
eq
iso
1
.2U (C) for methylene H atoms.
eq
Crystallographic data for the structure 3b have been deposited with the Cambridge
Crystallographic Data Center (CCDC) under the number 978361.
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Structure activity relationship
The structure activity relationship of the synthesized compounds has been analyzed on the basis
of the nature and the position of the substituents on the aromatic ring attached to the pyrrolone
ring.
•
Compounds having three methoxy groups at 3, 4 and 5-position of arylidene moiety were
found to have better anti-inflammatory activity as compared to those having one, two or
no methoxy groups.
•
Compounds having more electronegative (halogen atom) groups on arylidene ring
showed better anti-inflammatory activity, i.e. decreasing the size of halogen atoms
(
F>Cl>Br) increases the anti-inflammatory activity in order of F>Cl>Br. Compound 3b
having fluoro atom at arylidene ring showed better anti-inflammatory activity as
compared to compounds 3c and 3d.
•
•
Compounds having electro negative atom like F, o-Cl on benzyl ring showed better anti-
inflammatory activity as compared to no substitutent on benzyl ring, that is activity
increases in order of ( F> o-Cl> H).
Compound 3b having more electro negative group (F atom) on both ring i.e. arylidene
ring and benzyl ring showed maximum activity.
Conclusion
In the present study a library of twenty four compounds have been synthesized out of which two
compounds, 3-(4-fluoro-benzylidene)-5-(4-fluoro-phenyl)-2-chloro-benzyl-2(3H)-pyrrolone (2b)
and 3-(4-fluoro-benzylidene)-5-(4-fluoro-phenyl)-fluoro-benzyl-2(3H)-pyrrolone (3b) exhibited
significant in vivo anti-inflammatory activity as well as analgesic activity. Same compounds
suppressed TNF-α level significantly as compared to the standard drug. These two compounds
did not cause any damage in stomach and liver. Futher more, compounds 3b and 2b suppressed
the protein expression of COX-2 and NF-κB significantly as compared to the standard drug
indomethacin. Therefore compounds 3b and 2b may be considered as promising candidates for
development of new and safer anti-inflammatory agents.
Supporting Information
The supporting data provided with this article.
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Acknowledgements
The authors thank Vice Chancellor, Dr. G. N. Qazi for providing necessary facilities to the
Department of Chemistry. The authors are also thankful to Dr. Javed Iqbal, Director, Institute of
Life Sciences (ILS), Hyderabad for providing necessary facilities and Dr. Parimal Misra for his
help in conducting the in vitro studies. Yakub Ali acknowledges HNF (Hamdard National
Fellowship) for providing financial assistance.
Conflict of Interest
The authors report no conflicts of interest.
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(
Aporosa lindleyana Bail bark extract. J Ethnopharmacol. (accepted)
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2
2. Deraedt R., Joughney S., Delevakee F., Falhour M. (1980) Release of prostaglandin E
and F in an algogenic reaction and its inhibition. Eur J Pharmacol;51:17-24.
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Scheme 1: Protocol for syntheses of title compounds
I
F
F
COOH
O
II
F
R1
O
O
III
R1
F
O
O
HN
IV
R2
F
R1
N
O
R2
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Scheme 1: (I) Anhydrous AlCl , succinic anhydride; (II) aryl aldehyde, Ac O, sodium acetate,
3
2
reflux; (III) dry benzene, benzylamine; (IV) 6 N HCl.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.