PCBS-J-Vol 6-2-2012 W

PhytoChem & BioSub Journal
Peer-reviewed research journal on Phytochemistry & Bioactives Substances ISSN 2170 - 1768
PCBS Journal
Volume 6 N 2 2012

ISSN 2170-1768
ISSN 2170 1768
PhytoChem & BioSub Journal (PCBS Journal) is a peer-reviewed research journal published by Phytochemistry & Organic Synthesis Laboratory. The PCBS Journal publishes innovative research papers, reviews, mini-reviews, short communications and technical notes that contribute significantly to further the scientific knowledge related to the field of Phytochemistry & Bioactives Substances (Medicinal Plants, Ethnopharmacology, Pharmacognosy, Phytochemistry, Natural products, Analytical Chemistry, Organic Synthesis, Medicinal Chemistry, Pharmaceutical Chemistry, Biochemistry, Computational Chemistry, Molecular Drug Design, Pharmaceutical Analysis, Pharmacy Practice, Quality Assurance, Microbiology, Bioactivity and Biotechnology of Pharmaceutical Interest ) It is essential that manuscripts submitted to PCBS Journal are subject to rapid peer review and are not previously published or under consideration for publication in another journal. Contributions in all areas at the interface of Chemistry, Pharmacy, Medicine and Biology are welcomed. Editor in Chief Pr Abdelkrim CHERITI Phytochemistry & Organic Synthesis Laboratory Co-Editor Dr Nasser BELBOUKHARI Bioactive Molecules & Chiral Separation Laboratory Bechar University, 08000 Bechar, Algeria Editorial Board
Afaxantidis J. (France), Akkal S. (Algeria), Al Hamel M. (Morocco), Al Hatab M. (Algeria), Aouf N. (Algeria), Asakawa Y. (Japan), Atmani A. (Morocco) , Awad Allah A.( Palestine) , Baalioumer A. (Algeria), Badjah A.Y. ( KSA), Balansard G. (France), Barkani M. (Algeria), Belkhiri A. (Algeria), Benachour D. (Algeria), Ben Ali Cherif N. (Algeria), Benayache F. (Algeria), Benayache S. (Algeria), Benharathe N. (Algeria), Benharref A. (Morocco), Bennaceur M. ( Algeria), Bensaid O. (Algeria), Bhalla A. ( India), Bnouham M. (Morocco), Bombarda E. (France), Bouchekara M. (Algeria), Boukir A. (Morocco), Berada M. ( Algeria), Bressy C. (France), Chehma A. (Algeria), Chemat F. (France), Chul Kang S. ( Korea), Cravoto G. ( Italia), Dadamoussa B. (Algeria), Daiche A. (France), Daoud K. ( Algeria), De la Guardia M. ( Brazilia), Dendoughi H. (Algeria), Derdour A. (Algeria), Djafri A. (Algeria), Djebar S. (Algeria), Dupuy N. ( France), El Abed D. (Algeria), EL Achouri M. (Morocco), Jouba M. (Turkey), Hacini S. (Algeria), Hadj Mahamed M. (Algeria), Halilat M. T. (Algeria), Hamed El Yahia A. ( KSA), Hamrouni A. ( Tunisia), Hania M. ( Palestine), Iqbal A. (Pakistan), Gaydou E. (France), Ghanmi M. (Morocco), Gharabli S. (Jordan), Gherraf N. ( Algeria), Ghezali S. (Algeria), Gouasmia A. (Algeria), Greche H. (Morocco), Kabouche Z. (Algeria), Kacimi S. (Algeria), Kajima J.M. (Algeria), Kaid-Harche M. (Algeria), Kessat A. (Morocco), Khelil-Oueld Hadj A. (Algeria), Lahreche M.B. (Algeria), Lanez T. (Algeria), Leghseir B. (Algeria), Mahiuo V. (France), Marongu B. ( Italia), Marouf A. (Algeria), Meklati F. (Algeria), Melhaoui A. ( Morocco), Merati N. (Algeria), Mesli A. ( Algeria), Mushfik M. ( India), Nefati M. (Tunisia), Ouahrani M. R. (Algeria), Oueld Hadj M.D. (Algeria), Pons J.M. ( France), Radi A. (Morocco), Rahmouni A. (Algeria), Raza Naqvi S. A. (Iran), Reza Moein M. (Iran), Rhouati S. (Algeria), Roussel C. (France), Roussis V. (Portugal), Saidi M. (Algeria), Salgueiro L.D (Portugal), Salvador J. A. (Spain),
Seghni L. (Algeria), Sharma S. ( India), Sidiqi S. K. ( India), Souri E. ( Turkey), Tabti B. (Algeria), Taleb S. (Algeria), Tazerouti F. (Algeria), Vantyune N. (France), Villemin D.
(France), Yayli N. (Turkey), Youcefi M. (Algeria), Zabut B. ( Palestine), Ziyyat A. (Morocco), Zouieche L. (Algeria)
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Peer-reviewed research journal on Phytochemistry & Bioactives Substances
ISSN 2170 - 1768
PCBS Journal
Volume 6 N 2
POSL
2012
Edition LPSO Phytochemistry & Organic Synthesis Laboratory http://www.pcbs.webs.com , Email: phytochem07@yahoo.fr
PhytoChem & BioSub Journal Vol. 6(2) 2012 ISSN 2170-1768

ISSN 2170-1768
2012 Vol. 6 No.2
Contents
PhytoChem & BioSub Journal Vol. 6 N 2
B. FASLA, F. Z. ZEGHADA, A. MAROUF & M. BENNACEUR
Cytotoxic and genotoxic effects of aqueous extracts of five Algerian medicinal plants on Allium cepa L. root tips
A. KEMASSI, A. OULD EL HADJ-KHELIL, Z. BOUAL, A. HAMID OUDJANA & M. D. OULD EL HADJ
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Activits biologiques des huiles essentielles brutes foliaires de Peganum harmala L. (Zygophyllaceae) sur les larves du cinquime stade et sur les adultes de Schistocerca gregaria (Forskl, 1775) (Orthoptera- Cyrtacanthacridinae)
M. CHENNI, S. NEGGAZ, Z. FORTAS & D. El ABED
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Etude du pouvoir antibactrien de lextrait mthanolique de la racine de Bryonia dioica Jacq., de la rgion dOran
S. RAHMANI, L.ZIANE, N. BELBOUKHARI & A. CHERITI
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The Saharan medicinal plant Limoniastrum feei: Ethnomedical survey and preliminary phytochemical screening of antibacterial extracts
H. DJERADI , A. KRALLAFA , A. BOUYACOUB & M. LAHRECHE
83
Etude thorique de la raction de Diels-Alder : Effet de substitution par un mtal de transition sur la cintique et la rgioslectivit daddition
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PhytoChem & BioSub Journal Vol. 6 (2) 2012 ISSN 2170-1768

ISSN21701768
2012 Vol. 6 No.2
Cytotoxic and genotoxic effects of aqueous extracts of five Algerian medicinal plants on Allium cepa L. root tips
B. FASLA*, F. Z. ZEGHADA, A. MAROUF & M. BENNACEUR
Laboratoire de Biochimie Vgtale et des Substances Naturelles Facult des Sciences, Dpartement de Biologie Universit dOran, 31000 Oran, Algrie
Received: January 04, 2012; Accepted: April 10, 2012 Corresponding author Email biofasla@hotmail.fr
Copyright 2012-POSL

The antimitotic and genotoxic effects of aqueous extracts of five medicinal plants: Tetraclinis articulata (Vahl) Masters; Withania frutescens Pauquy; Peganum harmala L.; Ruta chalepensis L. and Haloxylon scoparium Pomel was evaluated using the Allium cepa assay. Onion bulbs were exposed to 0.25; 0.50; 0.75 and 1% concentrations (m/v) of each extracts for microscopic analyses. The extracts of P. harmala and H. scoparium shown mitodepressive effects on cell division at the 1% concentration. All the aqueous extracts induce an important aberrations due to different mechanisms: inhibitory, aneugenic and clastogenic effects of the aqueous extracts on Allium cepa meristematic cells. The antigenotoxic effect observed on cells treated by sodium azide and then by the aqueous extract of leaves of Withania frutescens at 1% concentration requires a chemical characterization of this plant and structural elucidation of the active principle. The qualitative analyses realized by thin layer chromatography (TLC) shows the presence of coumarins and flavonoides in some active extracts. The quantitative analyses realized by spectrophotometric assay indicate an important level of polyphenols and flavonoids in the aqueous extracts of T. articulata and H. scoparium. Key words: Allium cepa L assay, Algerian medicinal plants, Antimitotic activity, Genotoxicity, Chromosomal aberration, Phytochemistry
INTRODUCTION The plants and their extracts were used for a long time in traditional medicine. Being given their biological properties, they are the primary sources of drugs employed for the treatment of various diseases. Approximately 2 to 4% of plant species contain a great quantity of antineoplasic and/or cytotoxic components (Petit et al., 1994). The compounds isolated from these species lead to the development of new powerful products which inhibit tumors development, like vinblastin and vincristin (alkaloid) isolated from Catharanthus roseus G. DON (Cragg et al., 2003).
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The meristematic cells plant constitutes a material of an incomparable quality for the study of the mitosis. They approach what one could callthe typical isolated cell or the fundamental unit (Olliviers, 1948). The use of the higher plants in bioessay appears to be an excellent indicator of the cytotoxic and genotoxic effects caused by various chemical or physical agents. These tests proved their great value since the plants express a great sensitivity to the mutagen agents and offer a good system of analysis of the genic and chromosomal aberrations in eukaryotic system (Grant, 1978). These bioassays are validated by the United Nations Environment Program (UNEP), the World Health Organization (WHO) and the United States Environmental Protection Agency (US EPA) (Grant, 1999). A. cepa is commonly used as a test for investigating environmental pollution factors (Fusconi et al., 2006), irradiation (Kovalchuk et al., 1998), waste water (Rank and Nielsen, 1994), osmotic stress (Radi et al., 2005), and evaluating anticancer potential by the first work realized by Levan (1938), studying the effect of the colchicine on the mitoses. Cytogenetically, A. cepa cells contain a reduced chromosomal number (2n=16); broad and long Chromosomes; high percentage of cellular division; easy detection of the chromosomal aberrations and easy coloring. On the handling plan, this test is relatively fast, vegetable material is available all the year and its easy to cultivate in the laboratory. In order to contribute to the valorization of the famous Algerian medicinal plants for their therapeutic virtues, this work is initially interested by the study of the antimitotic and genotoxic activity of five aqueous extracts of plants at different concentrations, followed by the study of the antigenotoxic activity of the aqueous extract of Withania frutescens Pauquy. on meristematic cells division of Allium cepa L. root tips MATERIAL AND METHODS Collection of medicinal plants The medicinal plants utilized in this study were selected on the basis of their ethnobotanical uses (Table 1) and their allelopatic activity (Zeghada et al 2009; unpublished). The botanical identification was made by Pr Abderrazak MAROUF, laboratory of plant Biochemistry and Natural Substances, Department of Biology, Faculty of Science, University of Oran, Algeria.
Table 1: Use of plants in traditional medicine.
Plant Tetraclinis articulata (Vahl) Masters Withania frutescens Pauquy
Family Cupressaceae
Part Leaves
Ethnobotanical uses Anti-inflamatory
Reference Tekaya-Karoui et al. (2006)
respiratory and Bellakhdar et al. (1991) intestinal infection hypotensive, hypoglycemic Ziyyat et al. (1997), Bnouham et al. (2002)
Solanaceae
Leaves Treatment of the Font Quer (1990) ulcers
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Peganum harmala L.
Zygophyllaceae
Seeds
Fever, diarrhea, Bellakhdar (1997) abortion, subcutaneous tumors and Rheumatic and Hammiche intestinal pains, (2006) diabetes and eczema. Di Stasi et al. (1994)
Maiza
Ruta chalepensis L. Haloxylon scoparium Pomel.
Rutaceae
Emmenagogue, Leaves abortive, antihelminthic, spasmolytic anti-inflammatory Treat the trouble anti-cancer activity
Atta and Alkofahi, (1998)
Chenopodiaceae Arial parts
sight Boukef, (1986) Sathiyamoorthy et al. (1999)
Preparation of plant extracts The plants are cleaned from all impurities (ground, parts died, parasitic plants, etc), dried in the drying oven at 50C during 24 h then crushed. Ten g of dried powdered material was extracted with distillated water by refluxing for three times during 30 mn .The extract was lyophilized and stored at - 20C. Five concentrations were prepared from the lyophilized extracts that were dissolved in synthetic medium (see A.cepa test) as follow: 0.25; 0.50; 0.75 and 1% Allium cepa test Onion bulbs Allium cepa L. (2n = 16) of the same size are obtained from local market. After removing brownish parts, the bulbs are properly washed by distilled water and placed in beakers containing 40 ml of a synthetic medium (CaSO4: 60; MgSO4 60; NaHCO4: 96; KCl: 4 Mg/L for a good culture) (Rank and Nielsen, 1997) during 72 h at ambient temperature of the laboratory (22C). The roots of A. cepa from 2 to 3 cm length are exposed during 24 h with the four increasing concentrations of the aqueous extracts (0.25; 0.5; 0.75 and 1%), at a rate of two bulbs per treatment. The synthetic medium is used as control (T) for experiment. Antigenotoxic activity of the aqueous extract of Withania frutescens Pauquy. The study of the antigenotoxic activity of the aqueous extract of Withania frutescens Pauquy. (leaves) on meristematic cells of A. cepa exposed to sodium azide (NaN3) increasing solutions (0.25; 0.50; 0.75 and 1%). NaN3 is classified as a very toxic chemical causing the inhibition of mitochondriale chain respiration and cellular death by apoptosis (Inomata and Tanaka, 2003). The experiment was carried out under the same operating conditions as those of the antimitotic activity, except that after a setting in culture in the synthetic medium during 72h, the twenty (20) cultivated bulbs are distributed as follows: 02 bulbs are cultivated in the
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synthetic medium and are taken as control (T); 02 bulbs are treated by the aqueous extract of Withania frutescens Pauquy. at 1% during 24h; 08 bulbs are treated by sodium azide (NaN3) with increasing solutions (0.25; 0.50; 0.75 and 1%) during 24h; 08 bulbs are exposed to sodium azide (NaN3) to the same concentrations during 24h, then treated by the aqueous extract of Withania frutescens Pauquy at 1% during the same period. Coloration The coloring of Feulgen requires initially hydrolysis of the root meristems in 1N HCl at 60C during 12 mn, in order to facilitate the fixing of the dye on the aldehydic groups released during this hydrolysis and to give thereafter a red coloring purplished to the chromosomes (Jahier , 1992). Slides preparation The hydrolyzed meristematic parts (approximately 2 mm) are isolated using a scalpel, deposited slide in a drop of 45% acetic carmin and crushed between slide and cover slide with gentle taping, in order to obtain a good spreading out of the cells. Microscopic evaluation All slides were coded and examined from right to left and top to bottom out using a microscope video - ZEISS type by using immersion oil at X 100 magnification. The cytogenetic analysis included: mitotic index (MI), cytotoxic limit value (CLV), phases index (PI) and index of aberrations (AI). The counting of the normal or aberrant cells is carried out on 6000 cells by concentration in extract at a rate of 600 cells per meristematic root tip, by taking into account all the phases of the cellular division: prophase (P), prometaphase (PM), metaphase (M), anaphase (A) and telophase (T). MI is defined by the ratio of cells stopped in mitoses on the total of the counted cells (6000) (Ikeda et al., 2000) according to the equation (1).
The cytotoxic limit value (CLV) was calculated from the MI of the treated sample/MI of the control) 100 according to Sharma (1983).
The PI is determined by the number of cells in prophase (or in prometaphase (PM), im metaphase (M), anaphase (A) and telophase (T)) on the number of cells examined (6000) multiplied by 100 (Gliska et al., 2007) according to the formula (3)
The frequency of aberrant cells (%) was calculated based on the number of aberrant cells per total cells scored at each concentration of each extract (Rcuciu and Creang, 2007) from the equation (4):
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The chromosomal aberrations met are various types: micronucleus (MNC); chromosomal bridge (B); chromosomal break (Br); late chromosome (L); chromosome with sticking aspect (S); binucleated cell (bnl cell); fragmentation (Frgt); pycnose (PCN), etc. Phytochemical analysis TLC Preliminary analysis of the five aqueous extracts by thin layer chromatography on silicagel F254 (Merck) were performed. The chromatograms were developed in different mobile phases for testing the presence of secondary metabolites (polyphenol, avonoids, tannins, alkaloids, coumarins, saponins, anthrones, anthranols, quinones, lignanes, sesquiterpenes lactones and triterpenes) as described by Wagner and Bladt (1996). The TLC plates were visualized under UV light at 254 and 365 nm. Determination of total polyphenol and avonoid content The polyphenol content of the five aqueous extracts was quantied by the FolinCiocalteu reagent and was expressed as gallic acid equivalents (Singleton et al., 1999). 100 L test samples were mixed with 500 L Folin Ciocalteaus phenol reagents. After 5 mn ofreaction on darkness, 1.5 mL Na2CO3 at 2% was added. These solutions were maintained at obscurity during 1 h and the absorbance was read at 720 nm. The estimation of avonoid content was carrying out according to the colorimetric method described by kim et al. (2003). 1500 L of distilled water was added to 500 L of every aqueous extract solution followed by the addition of 150 L of NaNO2 at 5 %. Aliquot of 150 L of AlCl3 (1:10 w/v) was added 5 min later. After 6 min, 500 L of NaOH 1 N was added and the absorbance was measured at 510 nm. Catechin was used as a standard for constructing a calibration curve. Spectrophotometric measurements were performed by using UV-Visible spectrophotometer 8500 (P Double BEAM). Statistical analysis The results are expressed in the form of average standard error (Means standard errors (S.E.M) calculated on the average from ten blades (for the cytogenetic study); the average of two or three experiments (for the proportioning of polyphenols and flavonoids). The experimental data are presented in the form of curves and histograms. Calculations were carried out using the software Microsoft EXCEL 2003. RESULTS Antimitotic and genotoxic activity Mitotic index and cytotoxic limit value
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Referring to the fig.3, the mitotic index of the meristematic cells of A. cepa increases considerably at the same level in cells treated by the aqueous extract of the leaves of T. articulata with all the concentrations tested. In the same case meristematic cells of A. cepa treated by the aqueous extract of the arial parts of H. scoparium Pomel. present a very important mitotic activity in particular with the concentrations 0.50% (71.71 0.22%) ; this activity decrease considerably with the concentrations 0.75 and 1% (22.60 0.24%; 6.75 0.13% respectively) compared to the control. The analysis of the cells treated by the aqueous extract of seeds of P. harmala L. during24 h reveals a remarkable reduction in the mitotic index of wich rate reached 18,38 0.09 % at the concentration 1% of the extract, i.e. reduction in half compared to the control (47.35 0.16%). The meristematic cells of A. cepa treated by the aqueous extract of the leaves of R. chalepensis present a slight mitotic index reduction at all the concentrations tested compared to that observed for the control.
Figure.3. Mitotic index of the meristematic cells of Allium cepa treated with aqueous extracts.
When the mitotic index decreases bellow 22% compared to the control it causes what we call lethal effect (Antonsie-wicz, 1990). A mitotic index reduction below 50% compared to the control is usually a subletal effect (Panda and Sahu, 1985) and is named limiting value of cytotoxicity (Sharma, 1983). According to these two definitions we will be able to deduce that the aqueous extract of seeds of P. harmala is regarded as sublethal for the cells of A. cepa at the concentration 1%, whereas with the same concentration the aqueous extract of the arial parts of H. scoparium is regarded as lethal. Phase indexes The phase indexes of the of A. cepa cells treated by the aqueous extract of T. articulata (leaves) is represented mainly by preprophases and prophases for all the concentrations tested (Fig. 4 a). The prophase represents the dominating stage, for both the control cells and the treated cells by W. frutescens extract (leaves). The other stages represent only few percent (Fig. 4 b). The proportion of the meristematic cells in division treated by the aqueous extract of P. harmala L. (seeds) and R. chalepensis (leaves) strongly decreases by increasing the concentrations (Fig. 4 c, fig.4 d). A strong phase
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indexes decrease was observed on cells treated by aqueous extract of H. scoparium (arial part) at 0.75 and 1% concentration.
Figure.4. Phase indexes of A. cepa meristematic cells treated by aqueous extracts
Aberration index The anomalies induced by the aqueous extract of T. articulata are chromosomal breaks (Cb) (Fig.5b) and sticky chromosomes (S) (Fig. 5 c), generally gathered in prophase (4.51%) and metaphase (0.31%) at the concentration 0.75%.
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The presence of micronucleus (MNC) and binucleated cells (bnl cell) was seldom observed. On the other hand we distinguishes the presence of what we called nuclear vacuums on cells at the prophase stage going from 1 to 5 vacuums per cell (Fig. 5 d), and whose rate increases with the concentrations tested. In addition, we noticed a thickening and a strong coloring of the prophasic chromatin thats the intensity increases also with the concentrations of the extract used (Fig. 5 e).
Figure.5. Some anomalies met on meristematic cells of A.. cepa treated by the aqueous extract of leaves of Tetraclinis articulata (Mast) Vahl. (Leaves) with increasing concentrations. a: overall picture of control cell; b: breaks in prophase; c: breaks in metaphase with sticking aspect; d: nuclear vacuum; e: thickening and intense coloring of prophasic chromatin. The most anomalies caused by the aqueous extract of W.frutescens are nuclear budding (4.68% at the concentration 0.50%) (Fig.6 a), micronucleus (MNC) (2.85 at the concentration 0.75%) (Fig. 6 b), breaks (B) (Fig. 6 c), sticky chromosomes (Fig. 6 d), late chromosomes (Ct) (Fig. 6 e), multipolar anaphases (AMP) (Fig. 6 f).
Figure 6. Cell structure and forms of changed mitoses in Allium cepa root tip cells following treatment by aqueous extract of. Withania frutescens Pauquy. (leaves) with increasing concentrations. a: cells with contracted nuclei; b: prophase with micronucleus; c: metaphase with micronucleus; d: breaks in anaphase; e: metaphase with sticking aspect; f: late chromosome in metaphase; g: multipolar anaphase; h: atypical cell.
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The cytogenetic analysis of cells treated by the aqueous extract of P. harmala indicates the presence of various types of anomalies which increase with the extract concentration. The majority of these are defined by the presence of pycnoses (PCN) (Fig. 7 a), binucleated cells (bnl cell) (Fig 7 b), sticky chromosomes (S) (Fig 7 c), breaks (B) (Fig 7 d); chromosomal bridges (Cb) (Fig 7 e); and late chromosomes (L) (Fig. 7 f). The multipolar anaphases (MPA) and micronucleus (MNC) are seldom noted.
Figure.7. Structure of meristematic cells of Allium cepa root tips after treatment of Peganum harmala seeds during 24h with increasing concentrations compared with the cells of the control (T) X 1000. a: chromosomes with sticking aspect in prophase; b: metaphase with sticking aspect; c: breaks in Anatelophase; e: chromosomal bridges in telophase; e: late chromosome in prophase; f and g: binucleated cell; h: pycnotic cells.
During the microscopic evaluation of the meristematic cells of A. cepa L. treated by the aqueous extract of R. chalepensis we noticed that the majority of the anomalies were observed mainly at interphase and prophase stages and that for all the concentrations tested. Cells in interphase coloring was hardly visible (Fig. 8 a). The prophase stage is characterized by the presence of cells where coloring is also hardly visible, and this for the concentrations 0.25% and 0.50% (Fig. 8 b). At the cells exposed to the concentrations 0.75% and 1%, we noticed that the prophases present a thickening nucleus accompanied by an intense coloring of chromatin (Fig. 8 c). With the four concentrations tested, the prophase stage is characterized either by the presence of total thickening of chromatin in certain cells, or by the presence of partial thickening chromatin (intense coloring) within the same cell; the not thickened part tending to disappear (very clear coloring or almost goes away) (Fig. 8 d). The presence of various sizes of cells was also observed for all the concentrations tested. These are of four types: cells with important cytoplasmic volume and a nucleus of reduced size (Fig. 8 e); cells with important cytoplasmic volume and a nucleus of an important size (Fig. 8 f); cells with very reduced cytoplasmic volume and a big nucleus (Fig. 8 g); cell where the nucleus diffuses in the cytoplasm, appearing to be coloured in magenta (Fig. 8 h). We noticed the presence of cells where the nucleus is completely disappeared that we named cells without nucleus (CWN) (Fig. 8 i)
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Figure.8. Some anomalies met on meristematic cells of Allium cepa L. caused by the aqueous extract of Ruta chalepensis L. (leaves) to increasing concentrations compared to the control (T) X 1000. a: cells in interphase with hardly visible coloring; b: prophases with a core thickened and an intense coloring of chromatin; c: prophases with hardly visible coloring; d: cells presenting a thickened part of chromatin and the other part tends to disappear; f: cells with important cytoplasmic volume and cells with reduced size of nucleus; g: cells with very reduced cytoplasmic volume with a bulky core; h: cells with nucleus diffusing in the cytoplasm; i: cells without nucleus; j: atypical cell.
The aqueous extract of H. scoparium caused a cytoplasmic fragmentation (Fig. 9 a) followed by a nucleus fragmentation too (Fig. 9 b). A budding nucleus (BN) (Fig. 9 c) and thickening of chromatin were noticed. The presence of cells without nucleus (CWN) was also observed for some cells.
Figure 9. Anomalies met on meristematic cells of Allium cepa L. treated by an aqueous extract of Haloxylon scoparium L. (arial parts) with increasing concentrations X 1000. a: cells with contracted nuclei; b and c: beginning of cytoplasmic fragmentation; d and e: beginning of nuclear fragmentation.
Antigenotoxic activity of the aqueous extract of Withania frutescens Pauquy. Meristematic cells control of A. cepa, shows an important mitotic index corresponding to 88.48 0.10% (fig 10). In the same way, the same cells of A. cepa treated by aqueous extract of W. frutescens at 1% exhibits an important mitotic activity corresponding to a value of 81.06 0.13%. A clear reduction of the mitotic index of the cells of A. cepa exposed to the sodium azide (NaN3) solutions for all the concentrations tested was noticed. But the cells exposed to sodium azide at 0.25% increased half once treated by the aqueous extract of W.
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frutescens with 1%, whereas we noticed a light increase for those exposed to the concentrations 0.50% of sodium azide then consequently treated concentration in extract of withania. In addition, we observed a strong mitotic reduction in the index for those exposed to the solutions of sodium azide with 0.75% and 1% then treated by the aqueous extract of W. frutescens with 1%.
Figure10. Antigenotoxic effect of Withania frutescens at 1% on A.cepa root tips exposed to sodium azide.
Phytochemical analysis TLC The preliminary phytochimical tests of the TLC carried out on the various studied extracts, made it possible to two groups of secondary metabolites: flavonoid and coumarins (Table 2). With regard to the other metabolites, there are two possibilities: either they miss, or their low content does not make it possible to detect them with the techniques used.
Table 2: Composition of the extracts in flavonoid and coumarins.
Plant T. articulata W. frutescens P. harmala
Part Leaves Leaves Seeds
Phytoconstituant N C 02 Coumarins 01 02 Flavonoid Coumarins 01 04 02 03
R.chalepensis Leaves Flavonoid H. scoparium Arial parts Coumarins
Rf 0.28 0.37 0.6 0.32 0.32 0.14 0.1 0.21 0.29 0.48 0.40 0.46 0.38 0.65 0.72
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Determination of total polyphenol and avonoid content The content polyphenols of the various extracts is, by descending order, as follows: T. articulata > H. scoparium > P. harmala > R. chalepensis > W. frutescens (fig. 11).
Figure.11. Total phenol contents (mg/g) of aqueous plants extracts. Data are given as means S.E.M. (n=3).
In the same way, the content of flavonodes is, by descending order: T. articulata > H. scoparium > W. frutescens > R. chalepensis > P. harmala (fig. 12).
Figure.12. Total flavonoid contents (mg/g) of aqueous plants extracts. Data are given as means S.E.M. (n=3).
DISCUSSION The mitotic index is regarded as a parameter making it possible to estimate the frequency of the cellular division (Marcano et al., 2004). The reduction in the mitotic activity of the meritematic cells of A cepa indicates a mitodepressif effect of the aqueous extract of P. harmala and of R. chalepensis for all the concentrations tested, as well as the aqueous extract of H. scoparium for the concentrations 0.75 and 1%. We could suggest that the tested extracts or their components must interfere with the normal development of the mitosis, by preventing a number of cells to enter in prophase and thus blocking the cycle mitotic lasting interphase (El-Ghamery et al, 2000).
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The reduction of the mitotic activity is probably due to the inhibition of the DNA and the nucleoproteins synthesis of the biological system (Chauhan et al., 1998), a prolongation of the duration of the S and G2 phases (Webster and Davidson, 1969), a modification or deterioration in the expression of certain genes Siddiqui et al. (2007). Increase in the number of preprophasic and prophasic cells of A. cepa treated by the aqueous extract of T. articulata let suggest according to DAmato (1954), that is due, either at long duration of treatment, or to the use of high amounts, which leads to the deceleration of the entry at other stages of the mitosis, in particular, metaphase, anaphase and telophase. A great number of prophase was observed in the meristematic cells of Pisum sativum L treated by cadmium at concentration of 250M after 18 hours of treatment leading to the mitotic index increase compared to the control cells (Fusconi et al., 2006). A lengthening of the cellular cycle with an extension of the G2 phase and stage prophase at the meristematic cells of A. cepa after treatment by cadmium at 30 M (Borboa and De la torre, 1996). In the same way, the high NaCl concentrations (150 mM), cause a remarkable increase in the number of prophases in the root tips of Centauria ragusina L. compared to the control after 10 days of experimentation (Radi et al., 2005). The increase in the number of prophase must probably be connected to an intense deterioration of microtubules by preventing the assembly of the chromosomes at the metaphase stage (Fusconi et al., 2006). The strange aspect of the short and thickened chromosomes observed in prophase may indicate the effect of the aqueous extract of T. articulata on the organization of the chromatin, which can be in relation to disorders in the quantity of the histones, or other proteins responsible for the control of the nuclear chromatin structure (Stryer, 1997). Similar changes in the structure of chromatin were observed on meristematic cells of A. cepa treated by the aqueous extract of the barks of Uncaria tomentosa Willd (Rubiaceae) (Kura et al., 2006), and the aqueous extract of the needles of Taxus baccata L. (Taxaceae) (Majewska et al., 2003). The rate of chromosomal breaks (B) met at the meristematic cells of A. cepa is considerably important at those treated by the aqueous extract of T. articulata compared to the other extracts tested. These chromosomal breaks are due probably to the clastogenic effects of the extract, and its action on the chromosomes is generally regarded as being due to an action on the ADN (Grant, 1978). Fiskesj (1993), suggests that the chromosomal breaks are associated to the formation of chromosomal fragments and the micronucleated cells. The presence of sticky chromosomes in prophase and metaphase is very important on the meristematic cells of A. cepa treated by the aqueous extract of P. harmala L. compared to other tested extracts. The sticking aspect of the chromosomes could be the result of the degradation or the depolymerization of the DNA (Darlington and Mc-leish, 1951) and a similar dissolution of the nucleoproteins (Kaufman, 1958), or of an intense contraction and condensation of the chromosomes (Ahmed and Grant, 1972). The change of consistency of the chromosomes which become sticking probably explains by the action of the phytochimic compounds of the aqueous extract of P. harmala L. on chromosomal proteins. The sticking aspect of the chromosomes reflects a high toxic effect, of an irreversible type usually and probably leads to cellular death (El- Ghamery et al., 2003). The late chromosomes present on the meristematic cells of A. cepa lets suggest that the aqueous extracts of W. frutescens, P. harmala and H. scoparium have clastogenic effects, resulting from a rearrangement of the chromosomal parts by a gain or a loss of one chromosome inside the genome (Panda and Panda., 2002). According to Gabara et al (2006), the late chromosomes or chromatides seems as consequence of malformation of the kinetochores, or a partial inactivation of the spindle division, driving with the prevention of their displacement to the other poles (Trkolu, 2008). These chromosomes are gradually
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gathered and can be surrounded by the nuclear membrane to form micronucleus (MNC) (ElGhamery et al., 2003). The frequency of micronucleus (MNC) is rather important on the meristematic cells of A. cepa treated by the aqueous extract of W. frutescens . This value reached 2.85% at the concentration 0.75% and whose majority is present more in prophase than in interphase. The micronucleus can be originating by the acentric fragments formed following chromosomal or chromatidic breaks (clastogenic answer) (Mller and Streffer, 1994), to the chromosomes late during the mitotic anaphase, or starting from the spindle dysfunctionment (aneugenic answer) (Grover and Kaur, 1999; Sudhakar et al., 2001). The presence of an important rate of binucleated cells (bnl cell) on the root tips of A. cepa treated by the aqueous extract of P. harmala (0.86% with the concentration 1%) was noted compared to the other extracts. Their presence lets suggest that they are due probably to the eruption of the process of the cytokinesis while acting on the formation of the phragmoplaste, and preventing the formation of the girls cells, leading to the appearance of polyploid cells (Grant, 1976). The binucleated cells are regarded as the result of an inhibition of the cytokinesis at any point of control of the cellular cycle (Ateeq et al., 2002). The microtubules are implied in the formation of the cellular plate, the extract of P. harmala must probably affect the microtubules by thus inhibiting the cytokinesis (Trkolu, 2008). A pycnose (PCN) is a variety of irreversible nuclear lesion testifying to cellular death; it is characterized by the extreme retraction of the nucleus which becomes hyper colorable (Roger, 2007). The rate of pycnotic cells met on the meristematic cells of A. cepa treated by the aqueous extract of P. harmala increases with the concentration to reach a maximum value of 13.8% with the concentration 1%. This lets suggest that this extract contains substances causing these nuclear lesions. The phenomenon of the nucleus which diffuses in the cytoplasm of the meristematic cells of A. cepa treated by the aqueous extract of R. chalepensis corresponds probably to a caryorrhexy, defined by the bursting of the nuclear mass (Roger, 2007). In addition, the cells treated by the same extract presenting a thickened part of chromatin (intense coloring) and the other part tends to disappear (coloring very clear or almost goes away) lead probably to the appearance of what we called cells without nucleus (CWN), probable result of a karyolysis. This one is defined like dissolution of the nucleus with loss of its tinctorial affinities (Roger, 2007). The phenomena of pycnose, caryorrhexie and karyolysis are accompanied by modifications of nucleo-proteins, with depolymerization of the DNA and hydrolysis under the effect of cellular enzymes (Roger, 2007). The cytoplasmic and the nucleic fragmentation (Fgmt) of the meristematic cells of A. cepa treated by the aqueous extract of H. scoparium lets suggest that is about necrosis. Necroses are cellular death known as accidental which occurs at the time of tissue damage and it implies groups of cells (Moreau, 2006). The cells where this process takes place have increasingly raised plodies since the DNA is duplicated in loop. Moreover, the sizes of the nucleus being increased, the volume of the cells follows this tendency. This mechanism could play a part in the ways of adaptation of the plants to the abiotic stresses. On the other hand, cells treated by the aqueous extract of R. chalepensis presenting reduced nucleus sizes think is acts probably about a aneuploidy. The aneuploidy characterizes a cell which, following a change, does not have the normal number of chromosomes. With regard to the study the antigenotoxic activity of the aqueous extract of Withania frutescens Pauquy. at 1%, it is probable that this extract carries on a antigenotoxic activity on the meristematic cells of A. cepa treated by sodium azide at the concentrations 0.25% from where the mitotic index increase at a value of 61,71% (almost half).
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In addition, the antigenotoxic activity of the aqueous extract of W. frutescens on the meristematic cells of A. cepa. treated by sodium azide with the concentrations 0.75% and 1% is null. These results suggest that the sodium azide was not eliminated from the cells from where strong reduction in the mitotic activity to the quoted concentrations. CONCLUSION The study of the antimitotic and genotoxic activity realized on the Allium cepa root tips shows that each extract tested acts according to a different mode of action. The presence of coumarins and the flavonodes in the extracts of these two plants could be responsable for the observed activity. The aberrations on the level of the mitotic phases would be the result of aneugenic effects of the substances contained in the extracts, causing a mitotic spindle dysfunctionment leading to chromosomal disorders lasting the cellular cycle and of clastogenic effects responsible for direct variable cytotoxic effects such as the breaks, or indirect, such as the inhibition of the synthesis of the enzymes or regulating proteins of the cellular division. The antigenotoxic effect observed on cells treated by sodium azide then by the aqueous extract of the leaves of W. frutescens arouses the interest to better characterize this plant chemically. REFERENCES Ahmed M. and Grant W.F., 1972. Cytological effects of the pesticides phosdrin and bladex in Tradescantia and Vicia faba. Can. J. Genet. Cytol., 14: 157165. Antonsie-wicz D., 1990. Analysis of the cell cycle in the root meristem of Allium cepa under the influence of the Ledakrin. Folia Histochemica et Cytobiologica, 28: 79-96. Ateeq B., Abul Ferah M., Niamat Ali M. and Ahmad W., 2002. Clastogenicity of pentachlorophenol, 2,4-D and butachlor evaluated by Allium root tip test. Mutation Research, 514: 105-113. Atta A.H. and Alkofahi A., 1998. Anti-nociceptive and anti-inflammatory effects of some Jordanian medicinal plants extracts. Journal of Ethnopharmacology, 28: 306311. Bellakhdar J., 1997. La pharmacope marocaine traditionnelle; Mdecine arabe et ancienne et savoirs populaires. Ibis Press. p.247. Bnouham M., Mekhfi H., Legssyer A. and Ziyyat A., 2004. Medicinal plants used in the treatment of diabetes in Morocco. Int. J. Diabetes & Metabolism, 10: 33-50. Borboa L. and De la Torre C., 2000. Adaptation to Cd (II) and Zn (II), and the caffeinepotentiated override of the G2 block induced by the checkpoint activated by DNA damage. Plant Biosyst., 134: 39. Boukef M. K., 1986. Les plantes dans la mdicine traditionelle tunisienne. Agence de Coopration Culturelle et Technique. p. 82-83. Chauhan L.K.S., Saxena P.N. and Gupta S. K., 1998. Cytogenetic effects of cypermethrin and fenvalerate on the root meristem cells of Allium cepa. Environmental and Experimental Botany, 42:181-189. Cragg G. M. and Newman D.J., 2005. Plants as a source of anti-cancer agents. Journal of Ethnopharmacology, 100: 7279. Curtay J.P. and Robin J.M., 2000. Intrt des complexes antioxydants. Centre dEtude et de Dveloppement de la Nutrithrapie, 1-4. DAmato F., 1954. Action des facteurs physiques et chimiques sur la mitose. Int. Botanique, 9 : 1-9.
67
Darlington C.D.and Mc-Leish L., 1951. Action of maleic hydrazide on the cell. Nature, 167 : 407-408. Di Stasi, L.C., Hiruma, C.A. and Guimaraes, C.M., 1994. Medicinal plants used in Brazilian Amazon. Fitoterapia 65: 529540. El-Ghamery A.A., El- Kholy M.A. and Yousser A., 2003. Evaluation of cytological effect of Zn2+ in relation to germination and root growth of nigella sativa L. and Triticum aestivum L. Mutation Reasearch, 537 (1): 29-41. El-Ghamery A.A., El-Nahas A.I. and Mansour M.M., 2000. The action of atrazine herbicide as an indicator of cell division on chromosomes and nucleic acid content in root meristems of Allium cepa and Vicia faba. Cytologia, 65 (3): 277-287. Fiskesj G., 1993. The Allium cepa in wastewater monitoring. Environ. Toxicol. Water., 8: 291298. Font Quer P., 1990. Plantas medicinales. El Dioscrides renovado. Barcelona. Labor. In Montes F.T., 2004. Nombres y usos tradicionales de las plantas silvestres en almera (estudio lingstico y etnogrfico). Instituto de Estudios Almerienses, ed., p. 352. Fusconi, A., Repetto, O., Bona, E., Massa, N., Gallo, C., Dumas-Gaudot, E. and Berta, G., 2006. Effects of cadmium on meristem activity and nucleus ploidy in roots of Pisum sativum L. cv. Frisson seedlings. Environmental and Experimental Botany, 58: 253260. Gabara B., Kalwinek J., Kozir A., akowska Z. and Brycki B., 2006. Influence of N, N-Bis (3-Aminopropylo) Dodecyloamine on the ultrastructure of nuclei in Aspergillus niger mycellium and on cell proliferation and mitotic disturbances in Allium cepa L. root meristem. Acta Biologica Cracoviensia Series Botanica, 48 (1): 45-52. Gliska S., Bartezak M., Oleksiak S., Wolska A., Gabara B., Posmyk M. and Janas K., 2007. Effects of anthocyanin-rich extract from red cabbage leaves on meristematic cells of Allium cepa L. roots treated with heavy metals. Ecotoxicology and Environmental safety, 68: 343-350. Grant W.F., 1978. Chromosome aberrations in plant as monitoring system. Environ. Health Perspect., 27: 3743. Grant W.F., 1999. Higher plant assays for the detection of chromosomal aberrations and gene mutations-a brief historical background on their use for screening and monitoring environmental chemicals. Mutat. Res., 426: 107112. Grover I.S. and Kaur S., 1999. Genotoxicity of wastewater samples from sewage and industrial effluent detected by the Allium root anaphase aberration and micronucleus assays. Mutat. Res., 426: 183-188. Hammiche V. and Maiza K., 2006. Traditional medicine in Central Sahara: Pharmacopoeia of Tassili Najjer. Journal of Ethnopharmacology, 105: 358367. Ikeda K., Pant B., Mishiro A., Ozawa K., Masujima T. and Sugiyama M., 2000. A convenient method for the evaluation of anti-tumor agents affecting the cell cycle. Journal of Bioscience and Bioenginering, 90 (5): 574-576. Jahier J., 1992. Techniques de cytogntique vgtale. INRA, ed., Paris. p. 181. Kaufman B.P., 1958. Cytochemical studies of changes induced in cellular materials by ionizing radiations. Ann. New York Acad. Sci., 59 : 553. Kim D.O., Chun O.K., Kim Y.J., Moon H.Y. and Lee C.Y., 2003. Quantification of polyphenolics and their antioxidant capacity in fresh plums. Journal of Agricultural and Food Chemistry, 51: 65096515. Kura M., Nowakowska J., Sliwinska E., Pilarski R., Ilasz R., Tykarska T., Zobel A. and Gulewicz K., 2006. Changes in chromosome structure, mitotic activity and nuclear DNA content from cells of Allium Test induced by bark water extract of Uncaria tomentosa (Willd.) DC. Journal of Ethnopharmacology, 107: 211221.
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Levan A., 1938. The effect of colchicine on root tip mitosis in Allium. Hereditas, 24: 471 486. Majewska, A., Furmanowa, M., Sliwinska, E., Gowniak, K., Guzewska, J., Kura M. and Zobel, A., 2003. Influence of extracts from shoots of Taxus baccata var. elegantissima on mitotic activity of meristematic cells of Allium cepa L. roots. Acta Societatis Botanicorum Poloniae, 63: 185192. Marcano L., Carruyo I., Fernndez Y., Montiel X. and Torrealba Z., 2006. Determination of vanadium accumulation in onion root cells (Allium cepa L.) and its correlation with toxicity. Biocell., 30: 259267. Moreau D., 2006. tude de nouvelles cibles molculaires de cancer bronchopulmonaire non petites cellules pharmacomodules par des substances originales naturelles et synthtiques. Thse Doc. Universit de Nantes. France. p. 321. Mller W.U. and Streffer C., 1994. Micronucleus assays. Adv. Mutugen. Res., 5 (1) : 129134. Olliviers H.R., 1948. Etude cytotoxicologique de linfluence de divers agents physiques et chimiques sur les plantules de Bl. Rev. Canad. Biol., 7: 35-159. Panda B.B. and Panda k.k., 2002. Genotoxicity and mutagenicity of metals in plants. In Prasad MNV and Strzalka K., 2002. , Physiology and biochemistry of metal toxicity and tolerance in plants. Kluwer. Academic Publishers. Ed., p. 395-414. Panda B.B. and Sahu U.K., 1985. Induction of abnormal spindle function and cytokenisis inhibition in mitotic cells of Allium cepa by the organophosphorus incecticide fensulfotion. Cytobios, 42: 147-155. Petit G.R., 1994. Marine animal and terrestrial plant anticancer constituents. Pure & App/. Chern., Vol. 66, Nos lO/ll, pp. 2271-2281. Rcuciu M. and Creang D., 2007. Cytogenetic changes induced bu aqueous ferrofluids in agricultural plants. Journal of Magnetism and Magnetic Materials, 311 : 288-290. Radi S., Prolic M., Pavlica M. and Pevalek-Kozlina B., 2005. Cytogenetic effects of osmotic stress on the root meristem cells of Centauria ragusina L. Environmental and experimental Botany, 54: 213 218. Rani G., Kaur K., Wadhwa R., Kaul S.C.., and Nagpal A. 2005. Evaluation of the antigenotoxicity of leaf extract of Ashwagandha Nagpal. Food and Chemical Toxicology, 43 : 9598. Rank J. and Nielsen M.H., 1997. Allium cepa anaphasetelophase root tip chromosome aberrations assay on N- methyl-N-nitrosurea, maleic hidrazide, sodium azide, and ethyl methane sulphate. Mutat. Res., 390 : 121127. Rank J., and Nielsen M.N., 1994. Evaluation of the Allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater. Mutation Research., 312: 17-24. Roger P., 2007. Smiologie anatomo-cinique des lsions vasculaires et circulatoires : Anatomie pathologique. A5 Lsions vasculaires et circulatoires. Facult de mdecine Montpellier Nmes. France. Sathiyamoorthy P., Lugasi-Evgi H., Schlesinger P., Kedar I., Gopas J., Pollack Y. and GolanGoldhirsh A., 1999. Pharm. Biol., 37: 188-195. Sharma C.B., 1983. Plant meristems as monitors of genetic toxicity of environmental chemicals. Curr. Sci., 52: 10001002. Siddiqui S., Meghvansi M.K. and Hasan Z., 2007. Cytogenetic changes induced by sodium azide (NaN3) on Trigonella foenum-graecum L. seeds. South African Journal of Botany, 73 : 632635. Singleton V.L., Orthofer R. and Lamuela-Raventos R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of FolinCiocalteu reagent. Methods in Enzymology, 299: 152178.
69
Stavric B. and Matula T.I., 1992. Flavonoids in Foods: Their significance for nutrition and health lipid soluble antioxidants. Biochemistry and Clinical Applications, 274-294. Stryer L., 1997. Biochemia. Wydawnictwo Naukowe PWN, Warszawa, p.10321069.In Kura et al., 2006. Sudhakar R., Ninge-Gowda K.N. and Venu G., 2001. Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia, 66: 235 239. Tekaya-karoui A., ben jannet H. and Mighri Z., 2006. Contribution ltude de la composition chimique des huiles essentielles des rameaux, tiges, cnes et racines de la plante Tetraclinis articulata poussant en Tunisie. International symposium on perfume, aromatic and medicinal plant : from production to valorisation . Tunisia. Trkolu S., 2008. Evaluation of genotoxic effects of sodium propionate, calcium propionate and potassium propionate on the root meristem cells of Allium cepa L. Food and Chemical Toxicology, xxx (2008) xxxxxx (Online Science Direct). Wagner H., and Bladt S., 1996. Plant drug analysis, a thin layer chromatography atlas. Springer-Verlag, ed., Berlin. 2nd edition. Webster P.L. and Davidson D., 1969. Changes in the duration of the mitotic cycle induced by colchicine and indol3ylacetic acid in Vicia faba roots. Journal of Experimental Botany, 20: 671-685. Ziyyat H., Legssyer A., Mekhfi H., Dassoili A., Serhrouchni M. and Benjelloun W., 1997. Phytotherapy of hypertension and diabetes in oriental Morocco. Journal of Ethnopharmacology, 58 : 45-54.
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ISSN 2170-1768
2012 Vol. 6 No.2
Activits biologiques des huiles essentielles brutes foliaires de Peganum harmala L. (Zygophyllaceae) sur les larves du cinquime stade et sur les adultes de Schistocerca gregaria (Forskl, 1775) (OrthopteraCyrtacanthacridinae)
A. KEMASSI, A. OULD EL HADJ-KHELIL, Z. BOUAL, A. HAMID OUDJANA & M. D. OULD EL HADJ
Laboratoire de Protection des cosystmes en Zones Arides et Semi arides Universit Kasdi Merbah-Ouargla, BP 511 Ouargla 30000 Algrie
Received: December 30, 2011; Accepted: April 04, 2012 Corresponding author Email akemassi@yahoo.fr
Copyright 2012-POSL
Ltude de la toxicit des huiles essentielles brutes foliaires de Peganum harmala L. rcolt Oued MZab dans la rgion de Ghardaa (Sahara septentrional Est algrien), sur les larves L5 et les individus adultes du Criquet plerin, laisse remarquer un effet toxique chez ce locuste du dsert. Aprs traitement, des larves du cinquime stade et des imagos S. gregaria, par les extraits bruts des huiles essentielles foliaires de P. harmala, des troubles de dsquilibres et des mouvements convulsifs sont observs. Ce sont les mmes symptmes nots, chez des insectes traits par les insecticides. Les temps ltaux 50 (TL50) valus aprs traitement, sont de lordre de 06 mn 12 pour les larves L5 et de 19 mn 21 pour les individus adultes de cet insecte. Les larves du cinquime stade de ce locuste du dsert, semblent plus sensibles laction des huiles essentielles brutes foliaires que les imagos. Mots cls: S. gregaria, Toxicit, P. harmala, Sahara, Huiles essentielles The biological activity of crude leaf essential oil of Peganum harmala L. harvested from Oued M'Zab in region of Ghardaa (Algerian septentrional Sahara), on the larvae L5 and adult individuals of desert locust, showed a toxic effect in the desert locust. After treatment, the fifth stage larvae and imagos of S. gregaria, by crude extracts of P. harmala leaf essential oils, problems of imbalances and convulsive movements are observed. These are the same symptoms noted, in insects treated with insecticides. The lethal time 50 (LT50) measured immediately after treatment, are of the order of 06 mn 12 ' of L5 larvae and for 19 mn 21' for imagos of this insect. The fifth stage larvae of the desert locust seem more sensitive to the action of essential oils as imagos. Keywords: S. gregaria, Toxicity, P. harmala, Sahara, Essential oils
1. Introduction En qute de nouvelles techniques pour protger les cultures contre les insectes nuisibles afin daugmenter la production agricole, pour une population mondiale sans cesse
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croissante, tout en prservant lenvironnement, les organismes et les institutions de recherches sorientent vers la lutte biologique (APPERT et DEUS, 1982 ; ANTHELME et al., 2006). La possibilit dutiliser des substances secondaires des plantes contre les insectes nuisibles en gnral et contre le Criquet plerin en particulier, a suscit beaucoup de travaux. Les plus rcents sont ceux de ABBASSI et al. (2003a, 2003b, 2004, 2005), OULD EL HADJ et al. (2006), ZOUITEN et al. (2006), IDRISSI et HERMAS (2008), DOUMANDJI-MITICHE et DOUMANDJI (2008), AMMAR et NCIR (2008) et KEMASSI et al. (2010). Le Sahara dispose dune biodiversit floristique exceptionnelle, constitue denviron 480 espces (MAIRE, 1933) dont on dnombre 162 espces endmiques dans le Sahara Septentrional seul et la quelle sajoute une tradition sculaire de pharmacope traditionnelle. Plusieurs espces sont connues par leurs proprits thrapeutiques remarquables (QUEZEL, 1963). Les plantes spontanes des zones arides sont considres comme lune des ressources phytogntiques qui prsentent un intrt agronomique, conomique, cologique mais aussi stratgique (UNESCO, 1960). Face ce constat, et pour mieux caractriser les potentialits de la flore saharienne et valoriser, la prsente tude recherche partir de Peganum harmala (Zygophyllaceae); une plante spontane du Sahara septentrional Est algrien, pargne par le Criquet du dsert, ses caractristiques acridicides. 2. Mthodologie de travail 2.1. Matriel biologique Le matriel biologique est constitu de larves du cinquime stade (L5) et d'imagos du Criquet plerin et des feuilles de Peganum harmala L., rcolt Oued MZab (rgion de Ghardaa Sahara septentrional Est algrien). 2.1. Elevage de Schistocerca gregaria Les larves du cinquime stade et les imagos du Criquet plerin expriments, sont issus d'un levage de masse maintenu au laboratoire de Protection des Ecosystmes en Zones Arides et Semi-arides du Dpartement des Sciences Agronomiques de lUniversit Kasdi Merbah-Ouargla. 2.1.2. Matriel vgtal Peganum harmala L. est une plante herbace vivace de la famille de Zygophyllaceae, tiges ordinairement peu rameuses, de 30 90 cm de haut, entrenuds assez courts. Elle prsente des feuilles allonges et irrgulirement divises en multiples lanires trs fines, fleurs blanches sales grandes avec des spales ingaux persistants qui dpassent la corolle et des ptales crmes lavs de rose-orang nervures jaunes, oblongs et subsymtriques. Cette plante pousse en Europe australe et austro-orientale, Asie mineure, Tibet, Iran, Turkestan, Syrie, Arabie, Egypte et en Afrique du Nord. En Algrie, P. harmala est commune aux hauts plateaux, au Sahara septentrional et mridional, et aux montagnes du Sahara central (MAIRE, 1933; OZENDA, 1991; U.I.C.N., 2001). Elle est utilise par les populations locales en fumigation pour traiter les convulsions des enfants; en dcoction et pommade pour le traitement des fivres et en frictions pour soigner les rhumatismes. P. harmala prsente des proprits anthelminthique, antipaludique, antispasmodique,
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enivrante et sudorifique. C'est une plante non broute par les animaux (OZENDA, 1991; UICN, 2001). 2.2. Extraction des huiles essentielles Les feuilles de P. harmala soumises lextraction sont prleves partir de plantules en stade vgtation, rcoltes de leur biotope dexistence naturelle loin des endroits anthropiss. A laide dun montage dhydrodistillation simple, les feuilles fraches de P. harmala, sont portes bullition pendant 6 heures, la dcantation est ensuite ralise. Le produit obtenu est dshydrat laide du sulfate de sodium anhydre, afin d'liminer le peu d'eau susceptible d'avoir t retenue dans la phase organique. Le produit ainsi obtenu, est une huile essentielle pure, servira pour le traitement des insectes. 2.3. Etude de la toxicit A laide dun micro-pulvrisateur (Ultra Bas Volume), les huiles essentielles pures, sont pulvrises directement sur les juvniles du cinquime stade et sur les adultes de S. gregaria afin dtudies leur action par contact. Il est not aprs traitement lactivit motrice et le taux ainsi que le temps de mortalit. Lexprimentation est suivie jusqu la mortalit de la totalit des individus des lots traits. A cet effet, 4 lots dinsectes raison de 60 individus dont 30 mles et 30 femelles par lot sont constitus, ce qui fait un total de 240 individus. Deux lots sont des larves du cinquime stade dont un pour le tmoin et lautre pour le traitement et, les deux autres sont constitus par des imagos avec un pour le tmoin et lautre pour le traitement. 2.4. Calcul du temps ltal 50 (TL50) Le temps ltal 50 (TL50), correspond au temps ncessaire pour que 50% des individus dune population, meurent suite un traitement par une substance quelconque. Il est calcul partir de la droite de rgression des probits correspondants au pourcentage de la mortalit corrige en fonction des logarithmes du temps de traitement. Il est utilis la formule de Schneider et la table des probits. Formule de Schneider: MC = [M2-M1/100-M1] x 100
MC: % de mortalit corrige; M2: % de mortalit dans la population traite; M1 : % de mortalit dans la population tmoin.
3. Rsultats 3.1. Effet des huiles essentielles de Peganum harmala sur la mortalit des adultes et des larves L5 de S. gregaria Au vu des rsultats des figures 1 et 2, il ressort que les huiles essentielles des P. harmala prsentent un effet ltal aussi bien sur les larves L5 que sur les adultes du Criquet plerin. Les larves du cinquime stade semblent plus sensibles que les adultes. Elles meurent les premires. Chez les larves L5, la mortalit est note partir de la troisime minute, aprs le
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traitement et 100% de mortalit est atteinte au bout de 08 mn 30, alors quelle est au bout de 30 mn 18 chez les adultes. Mais les premiers cas de mortalit sont perceptibles partir de la vingt-deuxime minute au dbut du traitement chez les adultes. En revanche, aucune mortalit nest enregistre chez les individus du lot tmoin et que toutes les larves L5 ont acheves leur mue imaginale au bout de 71 jours. Par ailleurs, il est important de souligner que les mles que ce soit leurs stades de dveloppement, larves L5 ou bien adultes semblent plus sensibles, elles meurent aussi tt que les femelles. En outre, des troubles dquilibre, des mouvements convulsifs, dfcation intense, perte de la capacit de se percher un support suite lincapacit de jointure tarsique, tremblements dappendices et accroissement de rythme respiratoire sont observs. Ces manifestations tmoignent laction neurotoxique et organohalogne des huiles essentielle de P. harmala sur le Criquet plerin qui est probablement la consquence de leffet de ses extraits vgtaux sur le systme nerveux des criquets. Laction neurotoxique des huiles essentielles de P. harmala sur les larves du cinquime stade et adulte du Criquet du dsert mane probablement de leffet des diffrents composs chimiques quelles constituent et en particulier les alcalodes sur le systme nerveux du Criquet du dsert (ABBASSI et al., 2005). Des manifestations analogues sont constates chez les criquets traits par les insecticides organohalognes sauvons utiliss en lutte contre les criquets essaimant (CHAUVIN, 1956; MORETEAU, 1991). Lexamen des cadavres des criquets traits sous la loupe binoculaire, fait ressorti linexistence des lsions au niveau de la cuticule, pour cela, les huiles essentielles de cette plante nexercent gure deffets sur la cuticule et leur action par inhalation peut tre envisage en raison des caractres volatiles des ses essences vgtales. ISMAN (2000) et CHIASSON et BELOIN (2007), en tudiant lactivit biologique des huiles essentielles de nombreuses plantes dont lorigan, basilic, marjolaine, thym, sauge, laurier, romarin, lavande et autres sur les Thrips, les Pucerons, les Aleurodes Coloptres et les Hymnoptres, notent que les huiles essentielles agissent directement sur la cuticule des insectes et acariens corps mou tel que les Thrips, les Pucerons, les Aleurodes et certains acariens. Par contre, elles sont avres moins efficaces sur les insectes cuticule dure tels que des Coloptres et Hymnoptres adultes et certains Acariens prdateurs. En revanche, il est noter galement que les individus mles que ce soit leur stade de dveloppement larves ou bien adultes meurent avant les femelles, cela est probablement li la diffrence du poids existant entre les mles et les femelles du Criquet plerin. Gnralement chez les locustes le dimorphisme sexuel est bien apparent, les individus mles psent moins comparativement aux femelles. Paralllement, il est admis que la rsistance aux toxiques diffre dune espce une autre, et pour la mme espce, elle est relative plusieurs facteurs dont le poids est un facteur essentiel. 3.2. Temps ltal 50 (TL50) des huiles essentielles de Peganum harmala L. sur les adultes et les larves L5 de Schistocerca gregaria Le tableau 1 et la figure 2 A, B regroupent les quations et les droites de rgression, les coefficients de rgressions et les valeurs de TL50 et TL90 valus pour les huiles essentielles de cette plante saharienne sur les larves L5 et les imagos du Criquet plerin. Lvaluation des temps ltaux 50 (TL50) et 90 (TL90) pour les huiles essentielles de P. harmala sur les larves L5 et adultes de S. gregaria, a permet de confirmer la rapidit daction de ces extraits sur les larves L5 comparativement aux adultes. Le TL50 rapport pour les larves du cinquime stade tant plus court, il est de lordre de 06 mn 12, quant pour celui valuer pour les adultes, est de 19 mn 21. Quant aux temps ltaux 90 (TL90) valus, ils sont de 07 mn. 56 et 41mn. 43 pour les larves du cinquime stade et les
74
adultes respectivement. Cela affirme la sensibilit des larves par apport aux adultes. Dailleurs, il est admis communment que la rsistance des insectes aux toxiques accrot en fonction du stade de dveloppement, et que les adultes sont gnralement plus rsistants que les larves (CHAUVIN, 1956).
Tableau 1- quation de rgression, coefficient de rgression et les TL50 et TL90 calculs pour les huiles essentielles de Peganum harmala
Stade
quation de rgression
Coefficient de rgression (R)
Temps ltal 90 Temps ltal 50 (TL 50) (minute) (TL 90) (minute)
Larve L5
y = 9,0624x 1,8647
0,9042
06 mn 12
07 mn. 56
Adulte
y = 3,8067x 2,8485
0,3542
19 mn 21
41mn. 43
Figure 1 Cintique de la mortalit cumule des larves L5 de S. gregaria tmoins et traites par les huiles essentielles de Peganum harmala
Figure 2 Cintique de la mortalit cumule des adultes de S. gregaria tmoins et traites par les huiles essentielles de Peganum harmala
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Huiles essentielles de P. harmala dans le temps sur les adultes de S. gregaria.
Huiles essentielles de P. harmala dans le temps sur les larves L5 de S. gregaria
Figure 2 (A, B) Action des huiles essentielles de Peganum harmala sur les larves du cinquime stade et sur les adultes Schistocerca gregaria dans le temps.
4. Conclusion Ltude de la toxicit des huiles essentielles de Peganum harmala sur les larves du cinquime stade et les adultes de Schistocerca gregaria mette en vidence leur pouvoir insecticide sur le Criquet plerin. Les larves du cinquime stade sont plus sensible laction biocide des huiles essentielles comparativement aux adultes, temps ltaux 50 (TL50) valu pour les larves L5 sont plus courts par apport ceux rapports pour les adultes de S. gregaria. Des symptmes de neurotoxicit sont rapports soit, des troubles de mouvement, mouvements convulsives, lincapacit de ce percher un support est galement nots chez les larves et les adultes exposs aux huiles essentielles de P. harmala, cela tmoigne leffet neurotoxique de ses extraits vgtaux sur le Criquet du dsert. Dans cette optique, lutilisation des huiles essentielles de P. harmala contre le Criquet plerin pourrait tre envisage. Ces composs naturels pourraient constitus un lment de base pour la synthse des nouvelles molcules efficacit particulire sur les acridiens et sans risque dintoxication environnementale. Nanmoins, au pralable, il est appropri daffiner les connaissances sur la composition chimique de cette essence vgtale, de ces proprits fonctionnelles puis den dterminer les modalits et possibilits dapplications sans nuire lcosystme et dans le respect de la sant humaine. 5. Rfrences bibliographiques
ABBASSI K., ATAY-KADIRI Z. and GHAOUT S., 2003a.- Biological effects of alkalods extracted from three plants of Moroccan arid areas on the desert locust. The Royal Entomological Society, Physiological Entomology, (28): 232-236. ABBASSI K., MERGAOUI L., ATAY KADIRI Z., STAMBOULI A. et GHAOUT S., 2003b.Effet des extraits de Peganum harmala L. (Zygophyllaceae) sur le Criquet plerin Schistocerca gregaria (Forskl, 1775). Zool. Baetica, vol. 13 et 14: 203-217.
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ABBASSI K., ATAY-KADIRI Z. et GHAOUT S., 2004.- Activit biologique des feuilles de Calotropis procera (AIT. R. BR.) sur le criquet plerin Schistocerca gregaria (Forskl, 1775). Zool. Baetica, vol. 15: 153-166. ABBASSI K., MERGAOUI L., ATAY-KADIRI Z., GHAOUT S. et STAMBOULI A., 2005.Activits biologiques des feuilles de Peganum harmala (zygophyllaceae) en floraison sur la mortalit et lactivit gnsique chez le criquet plerin. Zool. Btica, vol.16: 31-46. AMMAR M. and NCIR S., 2008.- Incorporation of Cestrum parquii (solonaceae) leaves in an artificial diet affected larval longevity and gut structure of the desert locust Schistocerca gregaria (Forskl, 1775). Tunisian journal of plant protection, vol. 3 (1): 27-34. ANTHELME F., WAZIRI MATO M., DE BOISSIEU D. et GIAZZI F., 2006.- Dgradation des ressources vgtales au contact des activits humaines et perspectives de conservation dans le massif de l'Ar (Sahara, Niger). La revue en sciences de l'environnement, vol. 7 (2): 1-12. APPERT J. et DEUS J. 1982.- Les ravageurs des cultures vivrires et marachres sous les tropiques. Ed. Maison neuve et Larose, Paris, 419 p. CHAUVIN R., 1956.- Physiologie des insectes. Le comportement, les grandes fonctions, cophysiologie. Ed. INRA., Paris, 917 p. CHIASSON H. et BELOIN N., 2007.- Les huiles essentielles, des biopesticides Nouveau genre. Revue de littrature "Antennae", vol. 14 (1): 3-6. DOUMANDJI-MITICHE B. et DOUMANDJI S., 2008.- Quelques agents biologiques susceptibles dtre utiliss en lutte anti-acridienne. Revue des rgions arides, vol. 3(21) :1154-1158. IDRISSI HASSANI L. M. et HERMAS J., 2008.- Effet de lalimentation en Peganum harmala L. (Zygophyllaceae) sur le tube digestif du criquet plerin Schistocerca gregaria Forsk. (Orthoptera, Acrididae). Zool. Baetica, vol.19: 71-84.ISMAN, M.B., 2000.- Plant essential oils for pest and disease management. Crop. Prot., vol. 9: 603-608. KEMASSI A., BOUAL Z., OULD EL HADJ-KHELIL A., DADI BOUHOUN M., OULD EL HADJ MD., 2010.- Activit biologique de lextrait dEuphorbia guyoniana (Boiss. & Reut.) (Euphorbiaceae) chez le Criquet plerin Schistocerca gregaria (Forskl, 1775) (OrthopteraAcrididae). Annales de Sciences et Technologie, Universit Kasdi Merbah- Ouargla, 2 (1): 60 MAIRE R., 1933.- tudes sur la flore et la vgtation du Sahara central. Mmoire de la socit d'histoire naturelle de l'Afrique du nord n3, Mission du Hoggar II, Alger, 361 p. MORETEAU B., 1991.- Etude de certains aspects de la physio-toxicologie d'insecticides de synthse chez le Criquet migrateur Locusta migratoria (R. & F.). La lutte antiacridienne. Ed. AUPEL-UREF, Paris: 167-178. OULD EL HADJ M. D., TANKARI DAN-BADJO A., HALOUANE F. et DOUMANDJI S., 2006- Toxicit compare des extraits de trois plantes acridifuges sur les larves du cinquime stade et sur les adultes de Schistocerca gregaria (Forskl, 1775) (OrthopteraCyrtacanthacridinae). Scheresse, vol.. 17(3): 407-414. OZENDA P., 1991.- Flore et vgtation du Sahara. 3me dition, CNRS, Paris: 662 p. QUEZEL P., 1963 - La vgtation au Sahara. Ed. Masson et Cie, Paris, 33 p. U.I.C.N., 2001.- Connaissance, Valorisation et Contrle de lUtilisation de la Flore Sauvage en Mdecine Traditionnelle (Plantes Mdicinales). Programme Union Internationale pour la Conservation de la Nature pour l'Afrique du Nord. Ministre de l'Agriculture Algrienne, 153 p UNESCO, 1960.- Les plantes mdicinales des rgions arides. Recherche sur les zones arides XIII. Ed. UNESCO, Rome, 97 p. ZOUITEN H., ABBASSI K., ATAY-KADIRI Z., MZARI M., EL MAHI M. And ESSASSI E. M., 2006.- Insecticidal activity of Solanum sodomaeum (solonaceae) extracts on Schistocerca gregaria (Forskl) larvae. Journal of orthoptera research, 15 (2):171-173.
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ISSN21701768
2012 Vol. 6 No.2
Etude du pouvoir antibactrien de lextrait mthanolique de la racine de Bryonia dioica Jacq., de la rgion dOran
M. CHENNI 1, S. NEGGAZ 2, Z. FORTAS 2 & D. El ABED 1
1 2
Laboratoire de Chimie Fine Laboratoire de Biologie des microorganismes et de Biotechnologie Facult des Sciences, Universit d 'Oran, Es-Snia, Oran, Algrie
Received: March 09, 2012; Accepted: June 17, 2012 Corresponding author Email douniazed2000@yahoo.fr
Copyright 2012-POSL

Le prsent travail porte sur lvaluation du pouvoir antibactrien de lextrait mthanolique de la racine de la Bryone (Bryonia dioica Jacq.) dnomme Fashira ou Querioua en arabe appartient la famille des Cucurbitaceae. Les rsultats des tests dactivit biologique raliss sur lextrait mthanolique avec six (06) espces bactriennes (Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus), par la mthode de diffusion en milieu solide, ont montr que la racine de la Bryone possde un large spectre daction sur toutes les espces bactriennes testes. Mots cls : Bryonia dioica Jacq., Cucurbitaceae, Extrait mthanolique, Activit antibactrienne.
1. Introduction La mdication par les plantes date depuis les temps les plus reculs. Actuellement, elle connait une recrudescence auprs des populations. Prs de 80% de la population africaine ont recours aux plantes pour se soigner et nont pas accs aux mdicaments dits modernes. Au cours de la dernire dcennie, les extraits des plantes mdicinales et aromatiques suscitent un grand intrt, face la mfiance accrue par lusage des produits chimiques, aussi bien dans le domaine thrapeutique que dans le domaine alimentaire. Lutilisation des plantes ou de leurs extraits pourrait constituer une excellente alternative aux problmes de rsistance des antimicrobiens [1,2]. Aussi, un grand intrt a t rserv ltude des microorganismes, tant de point de vue biologique, que de point thrapeutique, suite lapparition de la rsistance des souches aux
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antibiotiques les plus communment utiliss et aux complications que ces germes produisent chez des patients profil clinique particulier [3,4]. Dans le cadre de laxe de recherche sur la valorisation de la biodiversit floristique algrienne et plus particulirement des plantes aromatiques et mdicinales, nous avons entrepris ltude microbiologique de la racine de la Bryone dioque dite Fashira ou Querioua, appartenant la famille des Cucurbitaceae [6]. En Algrie, cette plante est commune partout dans les rgions du Tell et plus rare ailleurs. Elle pousse parmi les broussailles et dans les forts [7]. Il est noter que dans louest Algrien particulirement Oran et Mascara, la racine sche de la Bryone est populairement appele Berestom par confusion avec celle du genre Aristolochia (A. baetica L. & A. paucinervis) qui appartient la famille des Aristolochiaceae. Elle est recommande pour le traitement de cancer sous forme de poudre avec de leau parfois mlange avec du miel en raison de son got amer. Cest un remde populaire utilis comme purgatif et dans le traitement de la goutte [8]. Elle est recommande essentiellement dans le cas des affections rhumatismales douloureuses et inflammatoires, tels que l'ulcre du duodnum, l'asthme, les bronchites et les pleursies [9]. Elle est prconise souvent dans le traitement de nombreux cancers (colon, abdomen, sein, peau, glande, etc.) tous les stades de la cancrogense [10]. Afin de justifier scientifiquement lutilisation de la Bryone en mdecine traditionnelle, nous nous sommes proposs de tester in vitro le pouvoir antibactrien de lextrait mthanolique de la racine de cette plante sur la croissance de diffrentes souches bactriennes. 2. Matriel et mthodes 2.1. Prparation de lextrait mthanolique Lextrait mthanolique test a t obtenu par extraction au soxhlet partir de la racine de la plante Bryone dioque rcolte dans la localit de Bouamama (rgion ouest dOran) en Octobre 2008 (Figure 1 et 2).
Figure 1: Origine de la Bryone 79
Aprs avoir rcupr la racine, nous lavons lave leau courante, dcoupe en rondelles (ou rouelles), puis sche au soleil et conserve temprature ambiante, dans un endroit sec et labri de la lumire (figure 3) [11]. Les rondelles sont ensuite broyes afin dobtenir une poudre fine, qui sera utilise dans nos exprimentations.
Figure 2 : Racine de la Bryone frache
Figure 3 : Racine de la Bryone sche en rondelles
2.2. Microorganismes tests Lactivit antimicrobienne de lextrait mthanolique de la racine de la Bryone dioque est teste sur six (06) souches bactriennes (tableau 1).
Tableau 1 : Souches bactriennes testes
Forme de sphre : Cocci Gram +
Souches bactriennes Enterococcus faecalis Staphylococcus aureus ATCC 6538
Provenance Institut Pasteur dOran Institut Pasteur dOran Institut Pasteur dOran Institut Pasteur dOran LBMB* Institut Pasteur dOran
Bacilles Bacillus subtilis ATCC 6633 Gram+ Forme de Escherichia coli ATCC25922 btonnet : Bacilles Gram - Proteus mirabilis ATCC 29906 Pseudomonas aeruginosa ATCC 14028
* Laboratoire de Biologie des Microorganismes et de Biotechnologie de lUniversit dOran Es-snia.
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2.3. Prparation des suspensions des microorganismes et incorporation au milieu de culture Les suspensions bactriennes sont prpares partir des pr-cultures de 24 heures, sur glose nutritive, dans 10 mL de bouillon nutritif et ajustes ltalon 0,5 Mac Farland (108UFC/mL). Aprs 24 heures dincubation 37C, 1mL dinoculum bactrien est mis dans chaque bote de Ptri contenant 20 mL de milieu Mueller-Hinton glos en surfusion. Trois disques de papier filtre de 6 mm de diamtre sont dposs dans chaque bote de Ptri, la surface du milieu ensemenc pour viter le chevauchement des zones dinhibition. Les disques sont pralablement imprgns de lextrait mthanolique dissout dans le mthanol 96% pour les cultures testes et uniquement de mthanol pour les cultures tmoins. Pour chaque souche bactrienne, nous avons effectu trois rptitions. Pour une bonne diffusion du contenu des disques, les botes sont laisses la temprature ambiante 15 mn avant de les incuber 37C. La sensibilit des souches bactriennes lextrait mthalonique est estime par la mesure des diamtres des zones dinhibition. Les cultures o il y a une croissance visible lil nu et suprieure 10 mm sont considres comme positives [12]. 3. Rsultats et discussion Les rsultats des tests, par la mthode de diffusion en milieu solide, sont rsums dans le tableau 1 et illustrs par lhistogramme de la figure 4 [13].
Tableau 2: Diamtres des zones dinhibition des bactries testes, aprs 24h dincubation 37C Souches bactriennes sensibles lextrait mthanolique
Staphylococ cus aureus ATCC 6538 Enterococcus faecalis Bacillus subtilis ATCC 6633 Escherichia coli ATCC25922 Pseudomonas aeruginosa Proteus mirabilis
14 mm
14 mm
12 mm
12 mm
10 mm
10 mm
Daprs les rsultats de lvaluation de lactivit antibactrienne, on constate que lensemble des souches bactriennes testes son sensibles laction inhibitrice de lextrait mthanolique de la racine de la Bryone. Les souches Staphylococcus aureus et Enterococcus faecalis sont les plus sensibles leffet de lextrait mthanolique. Alors que Escherichia coli et Bacillus subtilis peuvent tre considres sensibles. Pseudomonas aeruginosa et Proteus mirabilis sont faiblement sensibles. Laction de lextrait mthanolique sur les microorganismes possde un effet trs important sur les bactries Gram+ et moindre sur les bactries Gram-. Cette diffrence dactivit est due la structure de la paroi de la cellule cible.
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Diamtre (mm)
16 14 12 10 8 6 4 2 0
Souches Bactriennes
Proteus mirablis Pseudomonas aeruginosa Bacillus subtilis Escherichia coli Enterococcus faecalis Staphylococcus aureus
Figure 4 : Effet de lextrait mthanolique de la Bryone sur la croissance des bactries testes
4. Conclusion Les rsultats de cette tude montrent clairement que lextrait mthanolique de la racine de la Bryone est dot dun large spectre dactivit inhibitrice sur les souches bactriennes testes et particulirement les bactries Gram+ comme Staphylococcus aureus et Enterococcus faecalis. Rfrences
[1] Organisation Mondiale de la Sant (OMS), Aide mmoire, N 134, rvis Mai 2003. [2] Pousset, J.L., Medecine Tropicale, 2006, 66, p.606-609. [3] Farnsworth, N. R., Akerele, O., Bingel, A.S., Soejarto, D.D., Guo, Z., Bulletin de lOrganisation mondiale de la Sant, 1986, 64(2), p.159-175. [4] Roux, D., Catier, O., Botanique, pharmacognosie, phytothrapie, 3me Ed. Porphyre, 2007, p.13. [5] Ouis, N., Mise en vidence, Extraction et Analyse de quelques principes actifs de lAnis, du Fenouil et du Persil, Mmoire de Magister, Universit d'Oran Es-Snia, 2004. [6] Ait Youcef, M., Brette, J. P., Plantes Mdicinales De Kabylie Ed. Ibis Press, 2006, p.75. [7] Quezel, P., Santa, S., Nouvelles Flore De LAlgrie et des Rgions Mridionales, Ed. CNRS, 1963, p.893. [8] Evans, W.C., A Text Book of Pharmacognosy, 14th Ed. WB Saunders Company Ltd., 24-28 Oval Road, London, 1997, p.44, 474, 489-94. [9] Youngken, W.H., Text Book of Pharmacognosy, 6th Ed. The Blakiston Division, McGraw Hill Book Company Inc., New-York, 1950, p.478. [10] Duke, A. J., DuCellier, J., Duke, K. P., Handbook of Medicinal Herbs, 2e Ed, CRC Press, 2002, p.621. [11] Delille, L., Les Plantes mdicinales dAlgrie, Ed. Berti, 2007, p.65. [12] Leite, S. P., Vieira, J. R., de Medeiros, P. L., Leite, R. M., de Menezes Lima, V. L., Xavier, H. S., de Oliveira Lima, E., Evid Based Complement Alternat Med, 2006, 3(2), p.261-265. [13] Joffin, J., Leyral, G., Microbiologie Technique, 1996, p.43.
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ISSN 2170-1768
2012 Vol. 6 No.2
The Saharan medicinal plant Limoniastrum feei: Ethnomedical survey and preliminary phytochemical screening of antibacterial extracts
S. RAHMANI 1,2, L.ZIANE 1, N. BELBOUKHARI 1,2 & A. CHERITI 1
1) 2)
Phytochemistry & Organic Synthesis Laboratory Bioactive Molecules and Chiral Separation Laboratory University of Bechar, 08000 Bechar, Algeria
Received: December 18, 2011; Accepted: March 30, 2012 Corresponding author Email nasro14@maktoob.com Copyright 2012-POSL
Limoniastrum feei (plumbagenaceae) a medicinal plant, used in Saharan ethnopharmacopeae to treat gastric tract, hepatit desorder and cought. The antibacterial extracts from leaves, stem and twig of this plant are screened for the principal classes of secondary metabolites, such as Alkaloids, Saponins, Terpenes, Tannins, Flavonoids, Steroids and Cardenolids. Key words: Limoniastrun feei, bioactive extract, phytochemical screening, ethnopharmacology, Sahara
Introduction Compounds such as quinine, morphine, aspirin (a natural product analog), digitoxin and many others, so natural products are so important to undertake research are that they can be a source of new compounds. [1]. A dozen potent drugs have been derived from plants including: derived diosgenin; reserpine and pilocarpine. Other natural products are metabolites from fungi, bacteria, algae, and marine organisms. So, the diversity of structures obtained and the different therapeutic activities shown for the natural products make that the isolation, identification, synthesis and biosynthesis of new natural compounds continue to be a field of enormous interest [2]. Natural products have made enormous contributions to human health and have served as an important source of drugs since ancient times and about half of the useful drugs today are derived from natural sources. One of the most efficient ways of finding new bioactive compounds is collecting data on the use of medicinal plants in traditional pharmacopeia. Nearly 50,000 species of higher plants have been used for traditional treatment of illness. In systems of traditional healing, major pharmaceutical drugs have been either derived from or patterned after compounds from biological diversity [3]. Algeria with its large area and diversified climate has a varied flora, which is a source of rich and abundant medical matter and, in particular, Sahara part constitutes an important reservoir
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of many plants which have not been investigated until today. Among this flora, Limoniastrum feei belonging to plumbagenaceae family is widely applied in traditional folk medicine. [4-8]. The aim of this study is to validate the ethnomedical use of the Saharan medicinal plant Limoniastrum feei and to evaluate the antibacterial extracts by phytochemical screening. In earlier works we have reported the antimicrobial activity of aerial part crude extracts from Limoniastrum feei [9] , and we have isolated flavonoids and saponins from the methanolic extract of leaves and stem part of this specie [10, 11] Plant materials and extraction Aerial parts of Limoniastrum feei were collected in March May 2005 from Boukais (Bechar district) south-western Algeria. A voucher specimen is deposited at the herbarium of Phytochemistry and Organic Synthesis Laboratory under CA99/14 All parts of limoniastrum feei (leaves stem and twig) were cut into small pieces and shade dried at room temperature for one week, individually ground to a fine powder and stored in airtight polythene bags protected from sunlight until use. The solvent extraction of plant powder is done by using soxhlet apparatus in reflux for 3 hours. The extract is evaporated in vacuo apparatus to obtain a residue for the phytochemical screening. Botanical description Limoniastrum feei (syn. Ceratolimon feei , Bubania Feei ) - Plumbagenaceae- ( Figure 1), known under the name vernacular "Melefet Khadem", is a perennial shrub with densely ramified stems at the base, rather small, not exceeding 40 cm.The leaves form a basal rosette; they are lanceolated, approximately 5 cm. Flowering starts in early spring (end of February) and continues until late spring in May with a nice purplish red color of flower. Ecologically this Saharan specie thrives particularly on the stony grounds of the djebels, its occurrence on gravellysandy wadi beds. [5, 12, 13].
Stem Leaves Twig
Stem
Twig Leaves
Figure 1: General morphology of Limoniastrum feei 84
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Ethnomedical survey The ethnomedical survey was conducted in Bechar district (Figure 2). The majority of the population consists of four important origins: Dewimen, Ouled djerir, Cheraga and Ksouri. The language of the inhabitants is Arabic and Berber. The people's main source of living in this region is farming [5, 6]
Figure 2: The location of Study region, Algeria.
The ethnomedical survey carried out during three months especially in Bechar town. The interviewing question include: Vernacular name, plant parts used (dried or fresh), medicinal use, preparation methods and daily dosage. The information is taken from aged people especially women and herbalist witch have interesting traditional knowledge about plants. The information collected from the survey on the traditional uses Limoniastrum feei are shown in the following figure.
90 80 70 60 50 40 30 20 10 0 gastric disorrders hepatit cough
Preparations are usually made by using the aerial parts of the plant boiled in hot water. The water preparation is applied in the treatment of gastric disorders, hepatitis and cough.
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Phytochemical screening of the antibacterial extracts In our previous studies conducted in the biological screening of Saharan medicinal plants ,we have found that methanolic and heptane extracts of the three parts of Limoniastrum feei (leaves, stem and twig) have an interesting antibacterial effects ( Table 1). Thus we think that carrying out a phytochemical screening on the bioactive extracts for this plant is necessary to conducting a future works on the isolation and identification of natural compounds. [6, 9]. Table 1: Selected antibacterial Extracts
Bacteria Parts of plant Leaves Twig Stem Escherichia coli Pseudomonas aeruginosa Enterococcus facalis Klebsiella pneumoniae Staphylococu aureus
MeOH MeOH MeOH
MeOH MeOH MeOH
MeOH MeOH _
MeOH MeOH Heptane
MeOH MeOH MeOH
The crude of Metenolic and Heptane extracts of the three parts of Limoniastrum feei were screened for the presence of Alkaloids, Saponins, Terpenes, Tannins, Flavonoids, Steroids and Cardenolids, by using standard procedures to identify the constituents as described in literature [14, 15]. The results of phytochemical screening were given in the Table 2. Table 2: Phytochemical screening of the Methanolic and heptane extracts from Limoniastrum feei
Leaves M Alkalods Saponins Terpenes Tanins Flavonods Flavonods aglycon Glycosids flavonods Steroids + + + + + + + Twigs M + + + + + + Stems M + + + + + H + + + + + + + +
Cardenolids + + (M: Methanol, H: Heptan , + : detected, - : not detected)
Phytochemical Screening based on tests of colouration and precipitation was undertaken by the bioactive extracts (Methanol and Heptan). The tests carried out on leaves, Twings and Stems show presence of Saponins, Terpens, Tannins, Flavonoids (free and glycosides) and 86
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Cardenolids. However we observed less presence of steroids and absence of Alkaloids. From the results it is evident that all parts of the Limoniastrum feei gave a positive test for a tested class of natural compounds whereas we noted the absence of Cardenolids in leaves and steroids in Twing and Stems. Conclusion Limoniastrum feei (plombagenaceae) has great importance due to its ethnobotanical place in Saharan traditional medicine, for treatment of particularly gastric disorders. The phytochemical screening of the antibacterial extracts indicates the presence of Saponins, Tannins, Flavonoids and Cardenolids. The presences of this class of compounds are in agreement of the observed antibacterial activities. Finally, Limoniastrum feei can be a potential source of useful drugs and further studies are required to isolate the pure active metabolites. References [1] Newman, D. J., Cragg, G. M. 2007, Journal of Natural Products, 70, 461. [2] Hostettmann, K., Potterat, O., Wolfender, J.-L. 1998, Chimia. 52, 10. [3] Bisset N. 1994, Herbal drugs and phytopharmaceuticals. A handbook for practice on a scientific basis. Ed. Medpharm. Sc. Publishers, Sttutgarts-CRC Press, Boca Raton. [4] Cheriti, A.; Belboukhari, N.; Hacini, S., 2004 , Iran. J. Pharm. Res, , 3(2), 51. [5] Cheriti A., 2000, , Plantes mdicinales de la rgion de Bechar, Sud ouest Algrie (Ethnopharmacologie) Rapport CRSTRA, Algeria. [6] a) Cheriti, A., Belboukhari, N.& Hacini, S., 2001. Annales Univ. de Bechar,1. 4. b) Belboukhari N., Cheriti A. and Roussel C. 2007, eCAM, 4(S1), 55 . [7] Cheriti, A., Belboukhari, N. ,Sekkoum, K.. & Hacini, S. 2006, J. Algerien des Regions Arides, 5, 7. [8] Belboukhari, N. and Cheriti A. 2008, Elec. J. Environ., Agron., Food Chem., 7(14), 2749. [9] Belboukhari N; Cheriti A, 2005, Asian Journal of Plant Sciences , 4(5) , 49 [10] Belboukhari N; Cheriti A, 2007, Research journal of phytochemistry, 74-78 [11] Belboukhari N; Cheriti A, 2009, Chemistry of Natural Compounds, 45(5). [12] a) Ozenda P., 1967, Flore du Sahara, , Ed CNRS, Paris., b) Chehma, A. 2006 Catalogue des plantes spontanes du Sahara septentrional Algrien, Ed. Dar Houda, [13] Bellakhdar, J., 1997, La pharmacope marocaine traditionnelle. Mdecine arabe ancienne et savoirs populaires. IBIS Press. [14] Trease, GE., Evans, W.C., Pharmacognsy, 1989, 11th Ed. Macmillian publishers, [15] Harborne, J.B., 1978, Phytochemical methods,3rd Ed., Chapman and Hall, London
87

ISSN 2170-1768
2012 Vol. 6 No.2
Etude thorique de la raction de Diels-Alder : Effet de substitution par un mtal de transition sur la cintique et la rgioslectivit daddition
H. DJERADI1 , A. KRALLAFA1 , A. BOUYACOUB1 & M. LAHRECHE2
1) Laboratoire de Chimie Physique Macromolculaire 2) Laboratoire de chimie organique appliqu Universit Es Senia dOran, Algrie
Received: March 24, 2012; Accepted: June 30, 2012 Corresponding author Email h.djeradi@yahoo.fr
Copyright 2012-POSL
Depuis sa dcouverte il y a 75 ans, la raction de Diels-Alder est devenue un des outils le plus puissant dans la synthse organique. Bien que la cycloaddition [4+2] substitu par une fonction carbonyle a t largement tudie, les ractions impliquant les mtaux de transition comme substituant ont t peine dcrite. Nous proposons dans ce travail une tude thorique base sur des calculs quanto-chimique, de la raction dune mole du butadine substitu en position 2 par un TiCl3 avec deux moles dacroline laide de la mthode B3LYP/6-31G*. Deux chemins ractionnels sont envisags. Une cycloaddition [4+2] seffectue en premier, suivie par une addition [1,4]. Soit laddition [1,4] prcde la cycloaddition [4+2]. Mots cls : Cycloaddition [4+2], Addition(1,4), Rgioselectivit, TiCl3, DFT
Introduction La raction de Diels-Alder appele la cycloaddition (4+2) fut mise au point en 1928 [1] par Otto Paul Hermann Diels et Kurt Alder DAllemagne. Ils ont reue conjointement le prix Nobel de chimie en 1950 pour avoir dcouvert et dvelopp cette raction , qui est devenue un des outils les plus important dans la synthse organique[2] . Une quantit norme de travaux exprimentaux et thoriques a t consacre la cycloaddition [4+2 ] entre un cisoid conjugu et un Alcne. Les rsultats de ces travaux ont montr une remarquable rgio et strospcificit menant la formation des cycles six chneaux avec le haut contrle de la strochimie [3]. Limportance de la raction de Diels-Alder vient du fait quelle est gnralisable aux alcne et aux dines fonctionnaliss. On peu ainsi accder aux cyclohexnes substitus qui constituent
88
une partie du squelette de nombreuses molcules complexes tels les strodes[4], les sucres[5] et autres[6] . Le mcanisme de la raction de Diels-Alder est bien connu. Cependant les effets de substitution sur la cintique et la rgioselectivit daddition le sont moins. Dans le but dtudier leffet de substituant TiCl3 en position 2 du butadine sur la cintique et lorientation de la cycloaddition (4+2) ainsi que sa comptitivit laddition (1,4), nous avons dtermin laide de la mthode B3LYP/6-31G* les chemins ractionnels des diffrents modes daddition de ces deux ractions . Mthode de calcul La gomtrie de tout les ractants, les tats de transition, les intermdiaires ractionnels et les produits ont t optimise avec la mthode DFT [7], utilisant la srie Gaussien(98)[9]. En employant des fonctions hybrides dchange de corrlation B3LYP[10], associes aux bases de fonction atomique de type Gaussien double-zeta contenant des orbitales de polarisation pour une approche incluant des mtaux de transition . La 6-31 G* a t ainsi choisi, sachant que la B3LYP /6-31G* a t avec succs utilis dans les ractions de cycloaddition [11]. Un calcul de frquence a t excute pour tous les points stationnaires pour vrifier les tats de transition. Rsultats Laddition dune mole du butadine substitu en position 2 par unTiCl3 avec deux moles dacroline peut se faire par deux chemins ractionnels (figure 1). Passant par une cycloaddition [4+2] suivi par une addition [1,4] ou bien laddition [1,4] seffectue en premier suivi par la cycloaddition[4+2].
4+2
(1,4)
(1,4)
4+2
Figure 1 : schma ractionnel des deux ractions tudis
89
Mais lorsque le dine substitu par un TiCl3 est en prsence dun seul quivalant dacroline dans ce cas nous avons une comptition entre la cycloaddition [4+2] et laddition (1,4). Nous tentons dtudier les deux ractions utilisant la mthode B3LYP/6-31G* fin de prdire le chemin ractionnel le plus probable. 1- Etude de la cycloaddition[4+2]
Figure 2 : schma ractionnel de la cycloaddition[4+2]
Nous avons optimis les ractifs (1, 2, 3) utiliss pour cette tude. Les principaux paramtres gomtriques sont reprsents dans la figure 3 (les distances en Angstrms, et les angles en degrs)
1 R=H C5C6=1.338 C2C3=1.468 C6C5CO -0.029 / cis 179.967 /trans
2 C1C2=1.348 C2C3=1.475 C3C4= 1.340 C1C2C3C4= 53.90
3 C1C2=1.345 C3C4=1.339 C1C2C3C4=179.780
Figure 3 : structure des ractifs
Lapproche des deux ractifs 1 et 2 passe par un minimum reprsentant un cluster caractris par la formation dune liaison entre le doublet de loxygne de lacroline et une orbitale (d) de latome Ti. Lnergie de stabilisation de ce cluster est estim 9.2 kcal/mol
Figure 4 : formation dun cluster
90
quand au structures des tats de transition optimiss ; peuvent mener au diffrents conformres, de tout les modes daddition dans le nombre est huit, selon la position du groupement TiCl3 par rapport la fonction carbonyle : une foie de mme cot et une foie de part et dautre ; et aussi par rapport la double liaison de la fonction carbonyle en a deux forme (Endo /Exo) qui sont prsents dans la figure 5 TS1(1,4 exo-cis) TS2(1,3 exo-cis) TS3(1,3 endo-trans)
TS4(1,3 exo-trans)
TS5(1,4 exo-trans)
TS6(1,4 endo-trans)
TS7(1,3 endo-cis)
TS8(1,4 endo-cis)
Figure 5 structures des tats de transition
91
Les rsultats de loptimisation sont donns dans le tableau 1 suivant :

Tableau 1 : Rsultats de loptimisation des tats de transitions
TS1 E# (kcal/mol) C1C2 () C2C3 () C3C4 () C4C5 () C5C6 () C1C6 () C2Ti () 17.7 1.389 1.423 1.383 2.210 1.383 2.377 2.019
TS2 17.5 1.390 1.422 1.381 2.257 1.392 2.324 2.008
TS3 15.7 1.404 1.430 1.364 2.683 1.392 2.066 1.999
TS4 15.2 1.403 1.430 1.350 2.096 1.390 2.248 2.096
TS5 12.2 1.386 1.423 1.383 2.176 1.392 2.461 2.008
TS6 11.9 1.404 1.433 1.363 2.799 1.390 2.039 2.000
TS7 1 .5 1.350 1.437 1.383 2.147 1.398 3.527 2.136
TS8 -1.8 1.381 1.452 1.350 3.291 1.390 2.248 2.096
Les rsultats doptimisation des produits sont regroups dans la (figure 6) et le tableau 2, les valeurs inscrites sont les nergies de ractions, exprims en kcal/mol et les longueurs de liaison donnes en Angstrm Pour toute les orientations obtenus la raction est exothermique.
Tableau 2 : Energie et parametres gometriques des produits obtenus
Pr1 Er kcal/mole C1C2 () C3C4 () C4C5 () C5C6 () C1C2 () C6C1 () C2Ti () C1C2C3C4 deg -39.6 1.519 1.352 1.508 1.562 1.546 1.547 1.998 125.5
Pr2 -40.2 1.521 1.351 1.509 1.541 1.544 1.539 2.006 124.7
Pr3 -41.1 1.522 1.352 1.510 1.544 1.531 1.539 2.006 124.7
Pr4 -41.2 1.520 1.352 1.508 1.534 1.543 1.549 2.007 124.8
Pr5 -41.7 1.518 1.354 1.506 1.550 1.545 1.559 1.995 125.4
Pr6 -42.2 1.521 1.353 1.509 1.534 1.531 1.550 2.004 124.6
Pr7 -43.9 1.522 1.354 1.507 1.536 1.634 1.548 2.003 124.9
Pr8 -44.3 1.522 1.353 1.507 1.532 1.545 1.538 2.002 124.9
92
2 3
6 4 5
2 3
1 4
6 5
Pr1 Pr2
2 3
1 4
6 5
2 3 4
6 5
Pr3Pr4
2 3
6 5
2 3
6 5
Pr5Pr6
1 2 3 4 6 5 2 3
6 5
Pr7Pr8
Figure 6 : gomtrie des produits
93
2- La raction daddition (1,4)
Figure 7 : schma ractionnel de laddition(1,4)
Ls rsultats de loptimisation pour laddition [1,4] sont reprsent dans la figure 8 les nergies inscrite se sont lnergie dactivation et lnergie de raction donne en kcal/mol
H#= 29.8 kcal/mol
Hr= -53.0 kcal/mol
Figure 8 : optimisation pour laddition [1,4]
Discussion Pour les deux ractions la cycloaddition[4+2] ainsi que laddition(1,4) nous avons la formation dun cluster caractris par la formation dune liaison entre latome Ti et le doublet de loxygne. Huit tats de transitions on t trouvs pour la cycloadditon [4+2], les deux tats de transition les plus bas sont caractrises par la formation de liaison (O,Ti) qui est lorigine de leurs stabilit . Dans tous les tats de transition la distance C(butadine) C(terminal de lacroline) est la plus courte. De point de vue cintique la cycloaddition [4+2] lemporte sur laddition [1,4] puisque lnergie de transition de cette dernire et plus haut en nergie. De point de vue thermodynamique le produit le plus stable est bien le produit de laddition (1,4) Nous regroupement les rsultats de cette tude sous forme d'un profile nergtique reprsent par la figure 9
94
29.80
17.7 30.0 00 -1.8 7.4 -9 2

Coordonnes de la raction
Raction principale de Diels-Alder : Etat de transition le plus haut Etat de transition le plus bas Raction secondaire de Diels-Alder -44.3 -53.02 -43.9
Figure 9 : profile nergtique de la cycloaddition[4+2] et laddition(1,4)
Conclusion : La mthode B3LYP/6-31G* donne de bonne nergie dactivation compar lexprience. Pour le butadine portant le substituant TiCl3 nous avons envisag plusieurs modes dapproches, les deux tats de transition les plus bas sont caractris par la formation dune liaison entre loxygne de lacroline et latome Ti qui est un acide de Lewis. La stabilisation de ces tats de transition est du la chlation, dans ltat de transition de loxygne de lacroline par llment de transition Ti. La raction de cycloaddition[4+2]est cintiquement favoris par rapport laddition (1,4) contrairement ltude thermodynamique qui favorise le produit de laddition (1,4) Rfrences [1] Breson, j. A. Tetrahedron 1992,48,3. [2] Breson, j. A. J. Braz. Chem. Soc., Vol. 15, No. 1, 22-27, 2004. [3] (a) Nicolaou, K. C.; Snyder, S. A. Proc. Natl. Acad. Sci. USA 2004, 101, 1192911936; (b) Corey, E. J. Angew. Chem., Int. Ed. 2002, 41, 16501667; (c) Tietze, L. F.; Modi, A. Med. Res. Rev. 2000, 20, 304322. [4] Primean J.L., Anderson R.C.etFraser-Reid B., J.Am.Chem.Soc.;105,5874(1983) [5] Willard P.G.et De Laszlo S.E..; J.Am.Chem.Soc.,50,3738(1985) [6] Parr, R.G.;Pearson, R.G. ; J.Am.Chem.Soc.1983,105,7512.
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[7]Parr,R.G.;Yang,W.Density Functional Theory of Atoms and Molecules; Oxford University:New York,1989. [8]M.J.Frisch,G.W.Trucks,H.B.Schlegel,G.E.Scuseria,M.A.Robb,J.R.Cheeseman,V.G.Zakrze wski,J.A.Montgomery,Jr.,R.E.Stratmann,J.C.Burant,S.Dapprich,J.M.Millam,A.D.Daniels, K.N.Kudin,M.C.Strain, O.Farkas, J.Tomasi, V.Barone, M.Cossi, R.Cammi, B.Mennucci, C.Pomelli, C.Adamo,S.Clifford, J.Ochterski, G.A.Petersson, P.Y.Ayala, q.Cui, K.Morokuma, D K Malick, A.D.Rabuck,K.Raghavachari, J.B; Foresman, J; Cioslowski, J.V; Ortiz, A.G.Baboul, B.B.Stefanv, G.Liu, A. Liashenko, P. Piskorz, I.Komaromi, R. Gomperts, R.L. martin, D.J. Fox, T.Keith, M.A. Al-Laham, C.Y. Peng, A; Nanayakkara, C. Gonzalez, M.Challacombe, P.M.W. Gill, B. Johnsn, W. Chen, M.W.Wng, J.L. Andres, C. Gonzalez, M. Head-Gordon, E.S.Replogle, J.A. Pople, GAUSSIAN 98, Revision A.7,Gaussien,Inc.,Pittsburgh PA,1998 [9] A. D. Becke, J. Chem. Phys., 1993, 98, 5648. [10] W.Koch, M. C. Holthausen A Chemists guide to density Functional theory
96

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Plant Tetraclinis articulata (Vahl) Masters Withania frutescens Pauquy

Ethnobotanical uses Anti-inflamatory

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Figure.4. Phase indexes of A. cepa meristematic cells treated by aqueous extracts

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Plant T. articulata W. frutescens P. harmala

Part Leaves Leaves Seeds

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R.chalepensis Leaves Flavonoid H. scoparium Arial parts Coumarins

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Huiles essentielles de P. harmala dans le temps sur les larves L5 de S. gregaria

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