‫تقرير نهائي لمشروع بحث‬
Research project final report /
Rapport final du projet de recherche
‫مستند إداري‬
Administrative Document
--------
Administrative information / ‫المعلومات اإلداري‬
:‫المرجع‬
-Project Title)‫عنوان المشروع(عربي وأجنبي‬
Etudes complémentaires : géographique, comportementale et écologique de la vipère
de montagne Montevipera bornmuelleri incluant l’extraction, la purification, la
toxicologie et les caractéristiques pharmacologiques des molécules bioactives de son
venin.
Further geographic, ecological and behavioral studies of the Lebanon mountain
viper Montevipera bornmuelleri including extraction, purification and
pharmacological and toxicological characterization of bioactive molecules of its
venom.
‫( بمةةا فيهةةا‬Montevipera bornmuelleri ‫ل فعةةن الل نان ة الج ل ة‬
. ‫المملن‬
‫والعالج‬
‫جغراف ة وبيئ ة وسةةلول‬
‫دراسة اسةةتامال‬
‫استخراج المواد الفعال حيو ا في سم هذه األفعن وتنقيتها وتأثيراتها السم‬
Principal Investigator - ‫الباحث الرئ سي‬
U.L., Faculty of Sciences
II-Fanar, B.P. 90656,
‫العنوان‬
Address
Professeur Souad
HRAOUI-BLOQUET
‫االسم والشهرة‬
Name& surname
1
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
.1
Jdeidet El Maten-Lebanon
sdbloquet@yahoo.com
‫العنوان االلكتروني‬
e-mail
03141793 mobile
01 692954 desk
04 912024 home
‫رقم الهاتف‬
Lebanese
University
Faculty of Sciences II,
Fanar-Lebanon
Professor
‫المؤسسة‬
Institution
‫الوظيفة‬
Telephone
Post
Co-investigators - ‫الباحثون المشارلون‬
‫العنوان االلكتروني‬
e-mail
zfajloun @ul.edu.lb
rsadek@aub.edu.lb
walidhleihel@usek.edu.lb
‫المؤسسة‬
‫االسم والشهرة‬
Institution
Name and surname
U.L. Faculty of Sciences III- Prof. Ziad FAJLOUN
Tripoli- Lebanon
AUB, Biology Department
Dr. Riyad SADEK
Beirut - Lebanon
Université Saint Esprit Kaslik Dr. Walid Hleihel
(USEK) Medical Faculty,
Jounieh-Lebanon
Duration and starting date of the research /‫للمشروع وتاريخ دء البحث‬
‫المدة التعاقد‬
/ Duration (year)‫المدة التعاقدية للمشروع‬
2 years
Starting /2012/03/01, renewal the
2013/04/02 for one more year
Starting date of the research /‫وتاريخ بدء البحث‬
Scientific Information/ ّ‫ المعلومات العلم‬.2
-Objectives‫الهدف‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
Learning about the geographic and habitat distribution of an endemic and
endangered species of Lebanon contributing to its conservation and to learning
about its vulnerability to future climatic changes and direct human impacts.
Telemetric study to learn more about its ecology.
The extraction, purification, pharmacological and toxicological characterization of
bioactive molecules of the snake’s venom will lead to developing treatments for
2
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
envenomation cases and therapeutic application.
The toxic effects will be investigated in vivo on laboratory animals in terms of its
effect on physiological functions of these animals especially in relation to cardiac
muscles, blood vessels and nervous system.
Achievements - ‫أالنجازات المحقق‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
The ecological part (field study) biogeography, distribution, density, habitat, biological
activities (reproduction, alimentary regime, threaten etc…) and telemetric study. This last
point need more study.
The Biochemical study of the venom is done. Some proteins are determined and isolated
(L-Amino Acid Oxidase (Mb-LAAO; Phospholipase A2 (Mb-PLA2) in purpose to
determine its effects on blood, on bacteria, on animal tissues etc…
The physiological studies give interesting results on lethality and hemorrhage. In the
present study, we have evaluated the lethal, hemorrhagic and necrotic activities of
Montevipera bornmuelleri fresh venom.
Perspectives - ‫آفاق البحث‬
-
-
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
No research is done to study venom and its application in Lebanon
This study will find applications to medical domain, like a therapeutic application
for some diseases
Karyotype and molecular study let us to recognize the phylogeny of this viper to
confer for this to study done in another species of vipers (Wuster and al., 2007;
young, 2007; Murphy and al., 2007; Stumpel and jogger, 2007) and to identifiy its
relation with others species of the region.
It is possible to use the results to help in treatment of detoxification and to
production of anti-venom
- Publications & Communications‫المنشورات والمساهمات في المؤتمرات‬
3
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Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
Congress CMH2 of Morocco (Marrakech, 2011)
(2 communications)
1- Etude écologique d’une vipère endémique des montagnes du Liban: Montevipera
bornmuelleri
Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad Fajloun4
2- LC-ESI-MS analysis and biological properties of the crude venom of
Montevipera bornmuelleri
Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad Fajloun4
NB: 1- oral presentation; 2- posters
International Congress of Herpetology: Vancouver (Canada) August
2012)
( 3 communications)
1-
Biogeography and ecology of the Lebanon Viper Montivipera bornmuelleri with
some notes on its venom.
Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad Fajloun4
2- Preliminary proteomic analysis antibacterial and anti aggregate activities of the
crude venom of the Lebanon Viper Montevipera bornmuelleri.
Claudine Accary1, Souad Hraoui-Bloquet2, Riyad Sadek3, Walid Hleihel4 , and Ziad
Fajloun1
3- Effects and characterization of Montevipera bornmuelleri venom in a mouse
model
Walid Hleihel1, Riyad Sadek2, Ziad Fajloun3 and Souad Hraoui-Bloquet4
NB: 1- oral presentation; 2- poster; 3- poster
20th LAAS International Science Conferences - Beirut - Lebanon 2014
(5 communications).
1- Effets du venin de Montevipera bornmuelleri sur certains organes de souris
causant leur mort.
Eliane Kossaifi1, Walid Hleihel2, Claudine Accary3, Riyad Sadek4, Ziad Fajloun5and
Souad Hraoui-Bloquet6*
4
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
2- Purification and Biological Activities of Phospholipase A2 (Mb-PLA2) From
the Venom of Montivipera bornmuelleri, a Lebanese Viper.
Claudine Accary1, Souad Hraoui-Bloquet2, Katia Haddad1, Riyad Sadek3, Jean Claude
Desfontis4, and Ziad Fajloun1
3- Identification and antibacterial activity of L-Amino Acid Oxidase (MbLAAO) from the venom of Montivipera bornmuelleri, a Viper from Lebanon
Mohamad Rima1, Claudine Accary1 , Katia Haddad1, Riyad Sadek2, Souad HraouiBloquet3, Jean Claude Desfontis4, Ziad Fajloun1
4- Exploration des propriétés biologiques et Analyse protéomique du venin du
serpent Montivipera bornmuelleri à la recherche des biomolécules d'intérêts.
Claudine Accary1, Souad Hraoui-Bloquet2, Monzer Hamze1, Riyad Sadek3, Walid
Hleihel4, Ziad Fajloun1.
5- Effect of Montivipera bornmuelleri's Snake Venom on Human Blood:
Coagulation Disorder and Hemolytic Activity.
Claudine Accary1, Mohamad Rima1, Walid Hleihel2, Jean Claude Desfontis3, Ziad
Fajloun1 and Souad Hraoui-Bloquet4
NB: 2 et 4 oral presentations; 1-3 et 5 posters
Papers published and submitted (2 published and 3 submitted)
1: Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad Fajloun4 , 2012. An
ecological study of the Lebanon Mountain viper Montivipera Bornmuelleri (Werner,
1898) with a preliminary biochemical characterisition of its venom. Lebanese Science
Journal. Vol. 13, No. 1, 2012; p 89-101.
2: Mohamad Rima, Claudine Accary, Katia Haddad, Riyad Sadek, Jean Claude
Desfontis, Ziad Fajloun and Souad Hraoui-Bloquet*, 2013. Identification of L-Amino
Acid Oxidase (Mb-LAAO) with antibacterial activity in the venom of Montivipera
bornmuelleri, a viper from Lebanon. Infections Disorders- Drug Targets, 2013, Vol.
13, No . 5 , 7pages.
3: Claudine Accary, Souad Hraoui-Bloquet, Monzer Hamze, Yassine Mallem, Fawaz El
Omar, Jean-Marc Sabatier, Jean Claude Desfontis and Ziad Fajloun. Protein content
analysis and antimicrobial activity of the crude venom of Montivipera bornmuelleri;
a viper from Lebanon.
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4102 ‫برنامج دعم البحوث العلمية‬
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Submitted in Infectious Disorders - Drug Targets Journal beginning 2014
4: Claudine Accary, Aziza Mantash, Assem Elkak, Yassine Mallem, Souad HraouiBloquet , Ziad Fajloun, Purification and biological activities of phospholipase A2
(Mb-PLA2) from the venom of Montivipera bornmuelleri, a Lebanese viper.
Submitted in Toxicon Journal, end 2013
5: Claudine Accary, M. Rima, A. Khouzahya, W. Hleihel, Riyad Sadek, Jean Claude
Desfontis, Ziad Fajloun and Souad Hraoui-Bloquet*: Effect of Montivipera
bornmuelleri's Snake Venom on Human Blood: Coagulation Disorder and Hemolytic
Activity. Submitted in Infectious Disorders on 2014.
Abstract -‫موجز عن نتائج البحث‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
The ecological study of M. bornmuelleri and its habitat have been yet determined. A
preliminary telemetric study using trans meters, give us some details concerning its
activity in Sannine Mountains (Lebanon).
A biochemical study of its venom using specific methods of analysis (chromatography
and spectrometry), give the presence of a dozen of bioactive molecules (toxins, enzymes
and others…). Many enzymatic molecules have been extracted from this venom. An
exploration of the biologics properties of the venom has been done demonstrating a non
negligible effect of the venom on inflammatory reactions on some bacteria also on
coagulation of blood and the blood vessels. The damages caused in vital organs (kidney,
liver, heart…) by intra muscular injections are many (inflammation, Hemorragy, necrosis
oedema etc…) causing the death of the animal if no administration of anti-venom.
A physiological: Our results indicated that the venom of this viper had an important
lethal activity and average hemorrhagic and necrotic activities (in the area of
subcutaneous injection of venom) with correlation between the venom doses and these
toxic effects.
‫توق ع الباحث‬
Professor Souad Hraoui-Bloquet
6
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
Final report /
Rapport Final
Warning / Avertissement
1. The final report must be limited to results directly related to the research project
supported by the Council excluding any other activity carried out by the investigator
otherwise there port will be rejected.
2. Appendices maybe added or attached to the report.
1. Le rapport final doit être limité aux résultats directement liés au projet de recherche
soutenu par le Conseil à l'exclusion de toute autre activité menée par le chercheur
sous peine de rejet.
2. Des annexes peuvent être ajoutées ou attachées au rapport.
1. Principal investigator / Chercheur principal
Nameand surname /
Nom et prénoms
Professeur Souad HRAOUI-BLOQUET
Institution of affiliation /
Institution d'affiliation
Université Libanaise, Faculty of Sciences II-Fanar, Lebanon
2. Title of the project as proposed in the original application/
Titre du projet tel qu'il a été proposé dans la demande originale
(English and French / Anglais et Français)
Etudes complémentaires : géographique, comportementale et écologique de la vipère de
montagne Montevipera bornmuelleri incluant l’extraction, la purification, la toxicologie
et les caractéristiques pharmacologiques des molécules bioactives de son venin.
Further geographic, ecological and behavioral studies of the Lebanon mountain viper
Montevipera bornmuelleri including extraction, purification and pharmacological and
toxicological characterization of bioactive molecules of its venom.
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4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
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3. Purpose of the project / Objectifs du projet
(1page)
The study is concentrate on a mountain viper found at high altitudes in
Lebanese Mountains. The study is done, to learn about this endangered species
(Montevipera bornmuelleri) of Lebanon, its geographic and habitat distribution
and contributing to its conservation in addition to learning about its
vulnerability to future climatic changes and its interaction with the natural as
well as the human environment.
Biochemical studies done to found applications for some components in
medical domain and to found ways to reduce potential envenomation and
devising treatments based on studying the effects of snake’s venom on different
tissues:
- The biochemical study will concentrate on the composition of the venom
and the time required for production of venom.
- The toxic effects will be investigated in vivo on laboratory animals in
terms of its effect on histological and physiological functions of these
animals especially in relation to vital organs: cardiac muscles, blood
vessels, nervous system, lungs, liver, kidney…..
- It is possible to use the results to help in treatment of detoxification and
the production of anti-venom and in other medical applications especially
in relation to various therapeutic uses.
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4102 ‫برنامج دعم البحوث العلمية‬
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4. Expected outputs / Résultats attendus
1 page max / 1 page au maximum
Our scientific team of searchers have the aim to complete these interesting
investigation in many domains concerning the use of different components of
the venom of M. bornmuelleri. Satisfied results are yet obtained in the 3
domains of our study (biogeography and ecology, biochemical, physiological
and therapeutic).
Many things had to be done concerning the therapeutic applications of the
venom in medical domain and also in domain of cosmetology
The composition of the crude venom and isolation of its components are in a
good way. Some components are yet isolated and purified. Their effects are
studied: On blood, blood vessels, bacteria, etc… giving interesting results. The
results will found applications in therapy of some deseases. They will have their
place in surgical domain, in regenerations of tissues and in cosmetology.
Determination of the venom effect on the mice’s various organs to assess its
interest and to estimate the global toxicity of the Montivipera bornmuelleri
venom in mice. Equally, works will also be carried out to explore the interest of
the same venom on the cardiovascular system (cardiomyocytes and blood
vessels). Further work could also be made to study the venom transcirptome,
analyzing the venomous gland of Montivipera bornmuelleri and different
mRNAs expressed in this structure.
We need more time to do all things we had proposed to do in this therapeutic
domain.
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Résultats obtenus / Obtained results
5 to 10 pages / 5 à 10 pages
Appendices can be added a the end of this document / Des annexes peuvent être ajoutées à
la fin de ce document
Introduction
The aim of this work is to make an ecological study of this endemic
species Montivipera bornmuelleri in Lebanon mountains with a biochemical
characterization of its venom to investigate the presence of toxins, main
enzyme families involved in the bleeding response and/or some other proteins
with distinct biological activities and to isolate and purified them.
Montivipera bornmuelleri is an endemic and viviparous species from
Lebanon mountains. It is found only at very high altitudes 1900 and 2100 m. Its
habitat is arid with scarce vegetation cover (hraoui-Bloquet et al., 2002;
Hraoui-Bloquet et al., 2011).
The Viperidae venoms are complex mixtures of proteins, nucleotides
and inorganic ions (Markland, 1998; Fry, 1999). These combinations confer a
formidable array of toxic properties on the venom. These include peptides and
polypeptides responsible for a variety of toxic properties including a
perturbation of the coagulation cascade, the normal haemostatic system and
tissue repair. Consequently, envenomation by these snakes may generally result
in persistent bleeding. These proteins can be grouped into a few major protein
families,
including
enzymes
(serine
proteinases,
Zn 2+-dependent
metalloproteases, and group II phospholipase A2 isoenzymes) and proteins with
no enzymatic activity (C-type lectin-like proteins and disintegrins) (Ménez,
2002; Juárez et al. 2004).
Venomous Viperidae snakes represent a source of efficient bioactive
components that have already led to the development of several new drugs.
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Grant Program for Scientific Research in Lebanon – 2012
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Results
A: Biogeography and ecology of the Lebanon Viper Montivipera
bornmuelleri with some notes on its venom.
Montivipera bornmuelleri is endemic to the Lebanese mountains and
found only at altitudes above 1800m (Hraoui-Bloquet et al. 2002). It is a
relatively small viper with an SVL (Snout vent length) around 60 cm. It is
viviparous delivering a maximum of 6 live young. M. bornmuelleri is found in
high-altitude rocky terrain characterized by sparse montane thorny and cushion
plants. It lives In Oyoun Al Simane area in the Sannine mountain in the Mount
Lebanon chain. Ecology and geographic distribution was described and yet
published (see Hraoui-Bloquet et al. 2002 in journal of the CNRS Libanais, in
Appendices).
Our work in the telemetric domain is in progress. We need some more time to
complete our observations. Transmeters was inserted in abdominal cavity of
seven vipers: males and females, in the beginning of spring (May). When
individuals emerge from hibernation. Our preliminary results show that
individuals hibernate in the top of hill and they go down in the valley during
their active period. They do not go move very far from their hibernation
locality. The field observations were done every 10 days (from May to
October) to study their activity.
B- The physiological study of this viper premise to establish the lethal
proprieties of the venom and its hemorrhagic effect. The lethal dose is 50
(DL50) after intra-venosus, intra peritoneal and intra-dermic injections. This
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4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
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study give hemorrhage and necrosis described in Bitis gabinica (Scott a. et
Sherman a. minton, 2001).
The physiological study was conduct on the lethality by intramuscular injection
of the crude venom. The LD50 is the dose that causes the death of 50% of
animals within 24 h. The determination of the hemorrhagic, necrotic and
procoagulant activities give interesting results. The minimum hemorrhagic dose
(MHD) was determined. The minimum hemorrhagic dose (MHD) is 200µg.
The venom exhibited strong hemorrhagic and necrotic activities. The estimation
of MHD and (MND) minimum necrotic dose, help to determinate the doses of
antivenom that neutralize the hemorrhagic and necrotic effects.
We have evaluated the lethal, hemorrhagic and necrotic activities of
Montevipera bornmuelleri fresh venom. Our results indicated that the venom
of this viper species by intramuscular injections, had an important lethal
activity and average hemorrhagic and necrotic activities (in the area of
subcutaneous injection of venom) with good correlation between the venom
doses and these toxic effects. The intramuscular median lethal dose was
estimated at 12.19 mg/kg. The minimum hemorrhagic and necrotic doses of
this viper venom were evaluated at 225 μg and 180 μg respectively.
Next to this first study we are studying the effects of lyophilized venom of M.
bornmuelleri to determine: Ataxy, tachycardy, defecation, tail movement… and
the coagulation dose of this venom in mice. And to evalue the lethal,
hemorrhagic and necrotic activities to compare the results obtained with fresh
venom and those obtained with lyophilized venom.
NB: A paper will be published concerning this part of the work.
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C. Impacts of the crude venom of M. bornmuelleri in some vital organs in
mice causing their death
The study describes irreversible histological changes and damages caused by the
intra-muscular diluted crude venom injected to the laboratory mice. We observed
many histological damages in the kidney, liver, and heart. The damages are:
congestion of blood vessels, haemorrhag, inflammation, edema, destruction of
the membrane of Bowman capsule, pycnotic nucleus, necrosis. …Destruction of
the hepatocytes, in the renal glomerulus, in proximal and distal tubules of the
kidney…..all this damages conduct to the death of the mouse. Those damages are
dependant doses.
NB: A paper will be published concerning this part of the work.
D. Biochemistry and immunology and therapy
D-1: Proteomic analysis
Viperidae snakes represent a source of efficient bioactive components that have
already led to the development of several new drugs. Here, we describe a first
proteomic analysis of the venom of Montevipera Bornmuelleri, one viper from
the Middle-East Mountains. Thus, we analyzed the content of the venom
through Liquid Chromatography coupled to Electrospray Ionisation Mass
Spectrometry (LC-ESI-MS) techniques and identified some bioactive
molecules included in the crude product. Using the sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins according
to their electrophoretic mobility (a function of the length of a polypeptide chain
and its charge), sephadex G-75 gel filtration for the elution of different
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4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
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molecular size components and LC-ESI-MS for the analysis of different
fractions collected, we have cumulated a large body of data on molecular
masses and retention times and demonstrated the presence of PLA2, serine
protease, metalloproteases, bradykinin peptides, C-type lectin toxins, and other.
The antibacterial activity of crude venom and different fractions collected after
Sephadex gel filtration were studied on three bacterial strains: Escherichia
coli, Pseudomonas aeruginosa and Staphylococcus aureus. The effect of
crude venom on bleeding and platelet aggregation was also studied on blood
and citrated plasma. However, a better antibiotic effect of the crude venom was
observed against the Staphylococcus aureus bacterial species. The anti
aggregate effect on human platelets is demonstrated by the venom of this
Lebanese snake and could be generally associated with various compounds
disrupting hemostasis and leading to hemolysis of blood. Finally, our work will
lead to development of an appropriate methodology for characterization of a
new therapeutic molecule of natural origin.
D-2: Identification of L-Amino Acid Oxidase (Mb-LAAO) with
antibacterial activity in the venom of Montivipera bornmuelleri, a Viper
from Lebanon
L-amino acid oxidase (LAAO) is a multifunctional enzyme that is able to
develop different activities including antibacterial activity. In this study, a novel
LAAO (Mb-LAAO) was isolated from the venom of M. bornmuelleri snake
using size exclusion chromatography followed by RP-HPLC and partially
characterized. However, the molecular weight of the Mb-LAAO determined by
ESI-MS and SDS-PAGE was 59 960.4 Da. After the enzymatic activity test
confirming its identity (transformation of L-leucine), the Mb-LAAO was
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4102 ‫برنامج دعم البحوث العلمية‬
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evaluated for its antibacterial activity against Gram-negative bacteria. It showed
a remarkable effect against M. morganii and K. pneumoniae. Moreover, no
cytotoxic activity was observed for Mb-LAAO against human erythrocytes
arguing for an exploration of its pharmaceutical interest.
D-3: Effect of the Montivipera bornmuelleri snake venom on human blood:
coagulation disorder and hemolytic activities
Viper’s venom is a source of bioactive compounds with pharmaceutical
interest. Here, we studied the impact of the venom of M. bornmulleri snake on
human blood. The pro-coagulant and anti-coagulant activities are analyzed, the
prothrombine time is way above the normal value indicating an anti-coagulant
activity whereas for the activated partial thromboplastine time, the high
concentration of the venom showed an anti-coagulant activity, but a procoagulant effect was occurred when the venom concentration decreases to 50
and 25μg/ml. hemolytic tests were performed in suspension and on blood agar
plate andv results showed that the increased concentration of the crude venom
going until 1.6 mg cannot produce a hemolytic effect, even in the presence of
Ca++. Also, on the blood agar plate no hemolytic area appeared even with 40
μg of the lyophilized venom. Otherwise, the venom was able to induce a low
hemolytic effect by acting on L-α phosphatidylcholine used as substrate.
D-4: purification and biological activities of phospholipase A2 (Mb-PLA2)
from the venom of Montevipera bornmuelleri, a Lebanese viper
phospholipase A2 (PLA2), a common toxic component of snake venom, which
is responsible of a wide spectrum of pharmacological effects
including
bactericidal, anticoagulant and hemolytic activities. Her, a phospholipase A2
named (Mb-PLA2) was purified from the venom of the Lebanese Montevipera
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4102 ‫برنامج دعم البحوث العلمية‬
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bornmuelleri snake by using tow purification steps procedures consisting of
size-exclusion chromatography on Biogel P60 and reverse hogh pressure liquid
chromatography on Restek Ultra II C18 column. the experimental determined
molecular weight of the Mb-PLA2 was 13650 Da, which was determined by
electrospray ionization mass spectrometry and SDS-PAGE electrophoresis. The
purified Mb-PLA2 has shown a pro-inflammatory activity , and was evaluated
for bactericidal activity against Escherichia coli, Pseudomonas aeruginosa and
Staphylococcus aureus. Mb-PLA2 showed strong antibacterial activity against
Escherichia coli
similar to that of chloramphenicol antibiotic control.
Moreover, the Mb-PlA2 exhibited hemolytic activity toward human
erythrocytes as well as an anticoagulant activity when evaluated on human
plasma
D-5: Proteins content analysis and antimicrobial activity of the venom of
Montevipera bornmuelleri , a viper from Lebanon
Viperidae snake venoms represent a source of efficient bioactive components
that have already led to the development of several new drugs. In this work, we
analysed the protein content of the Montevipera bornmuelleri venom using LCESI-MS, sephadex G75 gel filtration and SDS-page and demonstrated the
presence of proteins with molecular masses corresponding to metalloprotese III,
serine protese and PLA2 in three fractions collected after filtration. Equally, we
examined the antimicrobial effect of the crude venom that showed an important
potency, as bactericidal agent, based on MBC and MIC values obtained, against
Staphylococcus aurrus and Morganella morganii bacteria. However, no
activity was registered against Enterococcus faecalis, being the most resistant
bacteria, neither against Aspergillus flavus and penicillium digitatum fungal.
Futhermore, on other bacterial strains and Candida albicans fungal tested, the
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4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
venom has shown an intermediate efficacy by slightly reducing their growth.
Our data concerning the M bormuelleri venom give evidence of a rich complex
content
aiding
the
exploration
of
few
bioactive
molecules
for
biopharmaceutical purposes.
5. Summary table of expected and obtained results/Tableau récapitulatif
des résultats attendus et des résultats obtenus
Expected
attendus
outputs
/
Résultats Obtained results / Résultats obtenus
17
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
1-Studies will be conducted in 1-The
venom
components
are
another locations (anti-Lebanon identified. Some bioactive molecules
and Hermont Mountain)
are determined (toxins, enzymes,
proteins…)
2-A closer look will be made on
the snakes activity patterns and 2-Some proteins identified and
movements in its environment (to purified and their action have been
complete the telemetric study) studied
and interactions with it.
3-Identification of L-Amino Acid
3-More venom components will Oxidase (Mb-LAAO) and antibacterial
be identified in the previous study activity tested
and will be isolated and purified
4-Proteomic analysis antibacterial and
4-Toxicology studies conducted anti aggregate activities of the crude
before using the whole venom will venom of the Lebanon Viper
be applied specifically to isolated Montevipera bornmuelleri are effected.
venom component. Regarding
their effects on cardiac, muscular
and nervous tissues and in the 5-Histologique and physiologique
future in therapeutic domains.
Effects of venom and damages caused
in liver, heart and kidney of the mice.
5-Determination of the venom
histologic effects on the mice : 6-Our results indicated that the venom
brain, cardiovascular system, of this snake species had an important
lungs, musles…will be do.
lethal
activity
and
average
hemorrhagic and necrotic activities (in
6- Further work could also be the area of subcutaneous injection of
made to study the venom venom) with good correlation between
transcirptome
analyzing the the venom doses and these toxic
venomous gland of Montivipera effects.
bornmuelleri
and
different
mRNAs
expressed
in
this 7- Une exploration des propriétés
structure.
biologiques du venin a été également
entreprise démontrant un effet non
7- Our purpose is to carry out the négligeable de ce venin sur les
extraction,
purification
and réactions
d’inflammation,
sur
characterization
of
new certaines souches bactériennes, sur la
compounds from the venom of coagulation sanguine et aussi sur les
the viper species Montivipera vaisseaux sanguins.
bornmuelleri.
Once
these
18
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
extracted bioactive molecules are
identified, we will optimize their
basic molecular structures by
detailed studies of structurefunction
relationships
using
synthetic structural analogues, in
order to achieve more selective
derivatives,
potentially
exploitable in therapy.
8- We study the lethal activity, the
average hemorrhagic and the necrotic
activities. The venom had an
important lethal activity, an average
hemorrhagic and necrotic activities (in
the area of subcutaneous injection of
venom) with good correlation between
the venom doses and these toxic
effects.
8-We will also identify and
synthesize
by
solid
phase
chemical inhibitors ligands antiAGEs
(Advanced
Glycation
Endproducts anti-AGEs) and
antioxidant substances from
natural origin. At this stage, a
preliminary study of the venom
was carried out and in parallel;
Solid Phase Peptide Synthesis
(SPPS) trial was performed to
elaborate
an
appropriate
synthesis protocol
9-we will study the effects of some
components on cancerous cells to
obtein a therapeutic applications
19
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
6. Possible encountered difficulties / Difficultés éventuelles rencontrées
No difficulty
- No scientific problems have been encountered
- Some studies had done in French laboratory (Nantes-France) by the
student in doctoral school (Mme Claudine Accary) because we have
not some specific products or equipments to do them in Lebanon.
- Some physiological studies are not do because no adequate
equipments in our laboratory
- The budget is not sufficient to cover all laboratory analyses and also
to bay all the chemicals products or some complementary equipment
we need for this study
7. Scientific publications )articles inpeer review journals, books,
communications, etc …) / Publications scientifiques (articles dans des
revues à comité de lecture, livres, communications, etc …)
Attach a copy of each publicationas it appeared in the journal)/ (Joindre une copie de
chaque publication telle qu'elle a paru dans la revue)
Papers published (2 published and 2 submeted)
1: Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad Fajloun4 , 2012. An
ecological study of the Lebanon Mountain viper Montivipera Bornmuelleri (Werner, 1898)
with a preliminary biochemical characterisition of its venom. Lebanese Science Journal.
Vol. 13, No. 1, 2012; p 89-101.
2: Mohamad Rima, Claudine Accary, Katia Haddad, Riyad Sadek, Jean Claude Desfontis,
Ziad Fajloun and Souad Hraoui-Bloquet*, 2013. Identification and Partial
Characterization of L-Amino Acid Oxidase (Mb-LAAO) From the Venom of Montivipera
bornmuelleri, a Viper From Lebanon. Infections Disorders- Drug Targets, 2013, Vol. 13,
No . 5 , 7pages.
3: Claudine Accary, M. Rima, A. Khouzahya, W. Hleihel, Riyad Sadek, Jean Claude
Desfontis, Ziad Fajloun and Souad Hraoui-Bloquet*, 2014: Effect of Montivipera
bornmuelleri's Snake Venom on Human Blood: Coagulation Disorder and Hemolytic
20
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
Activity. submited on 2014 for a specilised journal.
4: Claudine Accary4, Mohamad Rima4 , Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid
Hleihel3, Ziad Fajloun4 , 2012 Exploration des propriétés biologiques et Analyse
protéomique du venin du serpent Montivipera bornmuelleri à la recherche des
biomolécules d'intérêts. submited on 2014. for a specialised journal
8. Oral presentations or posters in national, regional and international
conferences / Présentations orales ou affichées à des congrès nationaux,
régionaux ou internationaux.
(Attach a copyof each presentationas it was presented orpublishedin refereedconference
proceedings)/(Joindre une copie de chaque présentation telle qu'elle a été affichée ou
publiée dans les comptes rendus des congrès)
Congress CMH2 of Morocco (Marrakech, 2011) international
(2 communications: one oral presentation , one poster)
1-
Etude écologique d’une vipère endémique des montagnes du Liban: Montevipera
bornmuelleri . Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad
Fajloun4. Oral presentation
2- LC-ESI-MS analysis and biological properties of the crude venom of Montevipera
bornmuelleri Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad
Fajloun4 . Poster
International Congress of Herpetology: Vancouver (Canada) August
2012)
( 3 communications)
1- Biogeography and ecology of the Lebanon Viper Montivipera bornmuelleri with some
notes on its venom. Souad Hraoui-Bloquet1*, Riyad Sadek2, Walid Hleihel3, Ziad
Fajloun4. Oral presentation
2-Preliminary proteomic analysis antibacterial and anti aggregate activities of the crude
venom of the Lebanon Viper Montevipera bornmuelleri.
Claudine Accary1, Riyad Sadek2, Walid Hleihel3 ,Ziad Fajloun1 and Souad HraouiBloquet4 . Oral presentation
3-Effects and characterization of Montevipera bornmuelleri venom in a mouse model.
21
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012
Walid Hleihel1 ,Claudine Accary2, Riyad Sadek3, and Ziad Fajloun2 , Souad HraouiBloquet4 . Poster
20th LAAS International Science Conferences - Beirut, Lebanon 2014
(5 communications).
1-Effets du venin de Montevipera bornmuelleri sur certains organes de souris causant leur
mort. Eliane Kossaifi1, Walid Hleihel2, Claudine Accary3, Ryiad Sadek4, Ziad Fajloun5,
Souad Hraoui-Bloquet6* . Poster
2-Purification and Biological Activities of Phospholipase A2 (Mb-PLA2) From the
Venom of Montivipera bornmuelleri, a Lebanese Viper'. Claudine Accary, Katia Haddad,
Riyad Sadek, Jean Claude Desfontis, Ziad Fajloun and Souad Hraoui-Bloquet6*. Poster
3-Identification and Partial Characterization of L-Amino Acid Oxidase (Mb-LAAO) From
the Venom of Montivipera bornmuelleri, a Viper From Lebanon'). Mohamad Rima,
Claudine Accary, Katia Haddad, Riyad Sadek, Jean Claude Desfontis, Ziad Fajloun and
Souad Hraoui-Bloquet*. Oral presentation
4-Exploration des propriétés biologiques et Analyse protéomique du venin du serpent
Montivipera bornmuelleri à la recherche des biomolécules d'intérêts. Claudine Accary,
Souad Hraoui-Bloquet, Monzer Hamze, Riyad Sadek, Walid Hleihel, Jean Claude
Desfontis, Ziad Fajloun. Poster
5-Effect of Montivipera bornmuelleri's Snake Venom on Human Blood: Coagulation
Disorder and Hemolytic Activity. Claudine Accary, Mohamad Rima, Achraf Kouzayha,
Walid Hleihel, Jean Claude Desfontis, Ziad Fajloun and Souad Hraoui-Bloquet. Poster
22
4102 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2012
Programme de subvention à la recherche scientifique au Liban – 2012